Becoming Taller with chondrocyte apoptosis

We know that chondrocyte apoptosis plays an active role in endochondral ossification.  When chondrocytes undergo apoptosis TGF-Beta may be released into earlier segments of the growth plate maintaining balance.  Apoptosis is an important part of height growth and in fact in fusing growth plates there were no signs found of apoptosis.  Chondrocyte apoptosis may also play a role in exerting force on the growth by water release from apoptotic cells.  When osteoclasts reabsorb the cartilage ECM, the swelling chondrocytes open.  The ECM contains a lot of water.  When the ECM is absorbed(the ECM is hydrophillic so it contains a lot of water) the reduction in water content outside the cell reduces leading to water being released out of the cell to restore equilibrium.  The force of this water being released may be what leads to bone deformation and you becoming taller.

Since fusing growth plates do not show signs of apoptosis and apoptosis may play a key role in the water release in order to make you taller it is clear that chondrocyte apoptosis is important to height growth.  Lack of chondrocyte apoptosis may be signs of senescence as chondrocyte apoptosis may release TGF-Beta in order to keep the height growth train chugging along.

Influence of stress magnitude on water loss and chondrocyte viability in impacted articular cartilage.

"Mature bovine cartilage explants were impacted with peak stresses ranging from 10 to 60 MPa at a stress rate of 350 MPa/s{This is huge, it's hard to get even 1 MPa with a method like LSJL}. Water loss, matrix axial deformation, dynamic impact modulus (DIM), and cell viability were measured immediately after impaction. The water loss through the articular surface (AS) was small and ranged from 1% to 6% with increasing peak stress{this is for the whole cartilage unit and not individual cells}. The corresponding axial strains ranged from 2.5% to 25%, respectively, while the DIM was 455.9 +/- 111.9 MPa. Chondrocyte death started at the articular surface and increased in depth to a maximum of 6% (70 microns) of the cartilage thickness at the highest stress{the deeper depth means more hydrostatic pressure since chondrocyte death stopped occurring at a depth of 6%, the chondrocyte death was likely not due to hydrostatic pressure}. The volumetric (axial) strain was more than twice the amount of water loss at the highest peak stress. Specimens impacted such that the interstitial water was forced through the deep zone (DZ) had less water loss, a higher DIM, and no cell death{So the cell damage could be due to the actual impact force and not to hydrostatic pressure as the region with the most hydrostatic had no cell death}. Matrix compaction in the superficial region [cause] higher compressive strains to occur at the surface rather than in the deeper zones."

Therefore, it is likely that very high impact force causes cell death rather than hydrostatic pressure.  Low levels of repeated impact may induce hydrostatic pressure via oscillatory interstitial fluid flow.  Hydrostatic pressure is likely to just increase the amount of water uptake by the chondrocytes leading to more force generated by chondrocyte apoptosis.

Increasing the osmolarity of joint irrigation solutions may avoid injury to cartilage: a pilot study.

"Saline (0.9%, 285 mOsm) and Hartmann's solution (255 mOsm) are two commonly used joint irrigation solutions that alter the extracellular osmolarity of in situ chondrocytes during articular surgery. [Does] varying the osmolarity of these solutions influences in situ chondrocyte death in mechanically injured articular cartilage{a higher osmolarity means more concentration of the substance per unit of water, so the higher the osmolarity the more water being released by the chondrocytes}? We initially exposed osteochondral tissue harvested from the metacarpophalangeal joints of 3-year-old cows to solutions of 0.9% saline and Hartmann's solution of different osmolarity (100-600 mOsm) for 2 minutes to allow in situ chondrocytes to respond to the altered osmotic environment. The full thickness of articular cartilage then was "injured" with a fresh scalpel. Using confocal laser scanning microscopy, in situ chondrocyte death at the injured cartilage edge was quantified spatially as a function of osmolarity at 2.5 hours. Increasing the osmolarity of 0.9% saline and Hartmann's solution to 600 mOsm decreased in situ chondrocyte death in the superficial zone of injured cartilage{so when more water was released from chondrocytes there was less chondrocyte death}. Compared with 0.9% saline, Hartmann's solution was associated with greater chondrocyte death in the superficial zone of injured cartilage, but not when the osmolarity of both solutions was increased to 600 mOsm."

So as the relative water outside the cell decreases, there is less chondrocyte apoptosis which is bad for the growth plates.

"The extracellular osmolarity experienced by in situ chondrocytes has not been measured directly. Cartilage (with high fixed negative charges) contains a high concentration of free cations (mainly Na+) and a low concentration of free anions (mainly Cl−) compared with surrounding synovial fluid, and its interstitial osmolarity is greater with precise values determined by the local proteoglycan concentrations and the Gibbs-Donnan equilibrium conditions"<-proteoglycan concentrations determine the osmolarity of the environment.  Growth plate chondrocytes don't have synovial fluid but neither do the chondrocytes in this study.

This study however shows a case where higher osmolarity levels resulted in more apoptosis.

Hyperosmotic stress-induced apoptotic signaling pathways in chondrocytes.

"Articular chondrocytes have a well-developed osmoregulatory system that enables cells to survive in a constantly changing osmotic environment. Osmotic loading exceeding that occurring under physiological conditions severely compromises chondrocyte function and leads to degenerative changes. [We] investigate the form of cell death and changes in apoptotic signaling pathways under hyperosmotic stress using a primary chondrocyte culture. A highly hyperosmotic medium (600 mOsm) severely reduced chondrocyte viability and led mainly to apoptotic cell death, while elevating osmotic pressure within the physiological range caused no changes compared to isosmotic conditions. A 600 mOsm hyperosmotic environment induced the activation of proapoptotic members of the mitogen-activated protein kinase family such as c-Jun N-terminal kinase (JNK) and p38, and led to an increased level of extracellular signal regulated kinase (ERK1/2). Hyperosmotic stress also induced the activation of caspase-3. In summary, our results show that hyperosmotic stress leads to mainly apoptotic cell death via the involvement of proapoptotic signaling pathways in a primary chondrocyte culture."

It could be the different properties of the fluids used in the two stuides that cause different results for example Saline contains a lot of positively charge ions.  Hartmann's solution is considered to be isotonic.  So at higher concentrations those two compounds may have had the effect of balancing the osmolarity rather than creating a hypo- or hyper- osmolarity.

"The interstitial osmolarity of cartilage ranges between 350 and 450 mOsm"

"Apoptosis leads to plasma membrane asymmetry and the externalization of phophatidylserine residues, which bind annexin V with high affinity. In the early stages of apoptosis, cells typically have an intact cell membrane. Thus, they do not stain with propidium iodide, whereas externalization of phophatidylserine can be detected by annexin V. In the late phase of apoptosis, cells stain with both dyes. The control group had more than 90% of intact, living cells and only less than 10% of cells in the early and late phases of apoptosis"

"The 400 mOsm-treated group showed no significant difference in the percentage of living and apoptotic cells compared to the control group. A marked, approximately 10-fold increase of apoptotic cells was observed in the 600 mOsm-treated group with a parallel decrease of viable cells"<-So an increase in osmolarity increases apoptosis.  Remember this study only occurs on chondrocytes so we don't know if there's more MSC differentiation into chondrocytes to compensate.  This is likely possible due to the release of TGF-Beta by apoptotic chondrocytes.

"Hyperosmotic stress leads mainly to apoptotic cell death that involves changes in the apoptotic signaling molecules in a primary chondrocyte cell culture. Hyperosmotic environment induced the activation of proapoptotic signaling factors such as JNK, p38 and caspase-3 and also led to an increased level of ERK1/2"

"Increasing osmotic pressure leads to a slight, transient change in cell growth, rate of protein synthesis and amino acid transport" 

The chondrocyte: a cell under pressure.

"The composition of cartilage reflects the net response of the chondrocytes to the prevailing [mechanical] loading pattern, with cartilage proteoglycan content highest in heavily loaded regions and removal of load leading to cartilage thinning and proteoglycan loss. Chondrocytes react to cartilage deformation and to the changes in hydrostatic pressure, extracellular ionic composition and streaming potentials induced by the load."

"When load is applied to cartilage, the tissue deforms as does the cell, leading to a rise in the hydrostatic pressure of the matrix within.  If the load is removed immediately, cartilage returns to its original conformation and pressure falls. However, cartilage is an osmotic system, and application of load disturbs the osmotic balance. Thus if load is maintained for any length of time, fluid is expressed in an attempt to restore osmotic equilibrium"

"Load deforms the matrix and chondrocyte, hydrostatic pressure rises, fluid is expressed increasing the extracellular concentration of proteoglycans and hence of cations."  Cell volume of the chondrocyte decreases.  Note we are trying to do this with the bone marrow and stem cells.  However, deformation of stem cells and bone marrow may lead to an increase in proteoglycans and differentiation into chondrocytes thus inducing height growth.  However, LSJL may delay chondrocyte apoptosis due to a reduction in cell volume when the growth plates are present.  However, it also increases extracellular concentration of proteoglycans leading to an eventual greater amount of water force expelled when the chondrocytes do undergo apoptosis.

Osmolarity affects chondrocyte apoptosis.  A high extracellular osmolarity may delay cellular apoptosis but may make the force of when apoptosis finally occurs stronger.

Some studies say that chondrocyte apoptosis is not necessary and chondrocytes can differentiate into pre-osteoblastic cells.  But either chondrocyte apoptosis is still important either to prevent it or to maximize the amount of water absorbed before apoptosis occurs.

Apoptosis may also be delayed by IGF-1 which is unusual considering IGF-1 increases chondrocyte hypertrophy(and other cells as well).  IGF-1 may delay apoptosis until hypertrophy is maximized.

Effect of exogenous IGF-1 on chondrocyte apoptosis in a rabbit intraarticular osteotomy model.

"Insulin-like growth factor-1 (IGF-1) [protects] chondrocytes from apoptosis in vitro. IGF-1 expression may also assist in maintaining a fully differentiated chondrocyte phenotype{Good for LSJL if we can find supplements to increase IGF-1, IGF-1 increases p-Akt which also inhibits apoptosis}. Administration of IGF-1 after fracture inhibits apoptosis in vivo. Twenty-four mature female New Zealand white rabbits were randomized to control and IGF-1 groups. All subjects underwent standardized medial femoral condyle fracture and repair. Fibrin clot was administered in all subjects, with 25 mcg/ml IGF-1 in the clot in half the subjects. Half of the animals in each group were sacrificed at 2 weeks and half at 4 weeks. Two-week controls showed significantly higher rate of apoptosis than 2-week IGF-1 subjects (21 +/- 6 vs. 12 +/- 6). Likewise, 4-week controls showed significantly higher rate of apoptosis than 2-week IGF-1 subjects{although we don't know if the IGF-1 subjects would eventually catch up or the IGF-1 hypertrophic cells differentiate directly into bone cells rather than undergoing apoptosis, in this study the 4 week IGF-1 don't show more apoptosis than 2 weeks but we don't know if this applies to the growth plates as this study involved an injury} (23 +/- 7 vs. 10 +/- 2). There was no significant administration difference between 2-week control and 4-week control subjects, or between 2-week IGF-1 and 4-week IGF-1 subjects. Intraarticular IGF-1 at the time of fracture repair appears to inhibit chondrocyte apoptosis in vivo, as judged by TUNEL staining, in this animal model. Administration of IGF-1 [may] inhibit human chondrocyte apoptosis in vivo"

"[IGF-1 stimulates] the addition of sulfated glycosaminoglycans (GAGs) to aggrecan molecules that will ultimately form the proteoglycans that make up much of the matrix of articular cartilage[and growth plate]. IGF-1 acts throughout skeletal immaturity to stimulate the proliferation of epiphyseal chondrocytes[this likely occurs indirectly through IGF-1's ECM stimulating properties] and thereby direct the linear growth of bones"<-IGF-1 stimulates ECM, ECM stimulates cellular proliferation and slows down chondrocyte apoptosis.

"During adulthood IGF-1 continues to regulate the homeostasis of articular cartilage by stimulating chondrocytes to produce GAGs and type II collagen. IGF-1 counteracts matrix degradation and chondrocyte apoptosis"<-this is true for growth plate cartilage too.

"Exogenous IGF-1 administration in an equine osteochondral defect model stimulates a response in chondrocytes implying that exogenous exposure of IGF-1 may extend and amplify endogenous IGF-1 response to articular injury"<-IGF-1 administration or indirect ways of increasing IGF-1 will amplify IGF-1 levels.

Since this study involves an injury a complete relationship cannot be drawn to growth plate chondrocytes but we know that IGF-1 inhibits(or delays) apoptosis.

Once chondrocytes have undergone autophagy or apoptosis they cannot dedifferentiate from bone into chondrocytes as they no longer exist.  This must be a key regulatory mechanism to prevent ectopic growth plate formation.

Fate of the hypertrophic chondrocyte: microenvironmental perspectives on apoptosis and survival in the epiphyseal growth plate.

"The terminally differentiated [hypertrophic chondrocyte] cells were considered to undergo a dramatic change in shape, size, and phenotype, and assume the characteristics of an osteoblast. While some studies have supported the notion of transdifferentiation, much of the evidence in favor of reprogramming epiphyseal chondrocytes is circumstantial and based on microscopic evaluation of cells that are present at the chondro-osseous junction. Although these investigations provided a novel perspective on endochondral bone formation, they were flawed by the failure to consider the importance of stem cells in osseous tissue formation. Subsequent studies indicated that many, if not all, of the cells of the cartilage plate die through the induction of apoptosis{if some cells of the cartilage plate undergo transdifferentiation then we can use those transdifferentiated cells that should maintain some of the epigenetic characteristics of epiphyseal chondrocytes to form new growth plates}. With respect to agents that mediate apoptosis, at the chondro-osseous junction, solubilization of mineral and hydrolysis of organic matrix constituents by septoclasts generates high local concentrations of ions, peptides, and glycans, and secreted matrix metalloproteins. Individually, and in combination, a number of these agents serve as potent chondrocyte apoptogens.  Hypertrophic cells [may] die through the induction of autophagy. In the cartilage microenvironment, combinations of local factors cause chondrocytes to express an initial survival phenotype and oxidize their own structural macromolecules to generate ATP. While delaying death, autophagy leads to a state in which cells are further sensitized to changes in the local microenvironment. One such change is similar to ischemia reperfusion injury, a condition that leads to tissue damage and cell death. In the growth cartilage, an immediate effect of this type of injury is sensitization to local apoptogens. These two concepts (type II programmed cell death and ischemia reperfusion injury) emphasize the importance of the local microenvironment, in particular pO(2), in directing chondrocyte survival and apoptosis."

"Pi uptake is required for activation of chondrocyte apoptosis, and specific Na-Pi transporters were identified in growth plate chondrocytes. When these transporters were inhibited, apoptosis was blocked. Ca2+ [is] a key cofactor in the induction of chondrocyte apoptosis"

The fate of the terminally differentiated chondrocyte: evidence for microenvironmental regulation of chondrocyte apoptosis.

"Chondrocytes contained within the epiphyseal growth plate promote rapid bone growth. To achieve growth, cells activate a maturation program that results in an increase in chondrocyte number and volume and elaboration of a mineralized matrix; subsequently, the matrix is resorbed and the terminally differentiated cells are deleted from the bone. The terminally differentiated epiphyseal cells are deleted from the cartilage by apoptosis. Indeed, morphological, biochemical, and end-labeling techniques confirm that death is through the apoptotic pathway. Since the induction of apoptosis is spatially and temporally linked to the removal of the cartilage matrix, current studies have examined the apoptogenic activity of Ca(2+)-, Pi-, and RGD-containing peptides of extracellular matrix proteins. All of these molecules are powerful apoptogens. With respect to the molecular mechanism of apoptosis, Pi [the] apoptogen anion is transported into the cytosol via a Na(+/)Pi transporter. Subsequently, there is activation of caspases, generation of NO, and a decrease in the thiol reserve. Specific microenvironments exist in cartilage that can serve to direct chondrocyte apoptosis."

"The most superficial region, closest to the joint surface, is the reserve or resting cell zone. In this region, spherical chondroprogenitor cells, embedded in an extensive extracellular matrix, are present. Below the reserve cell zone is the proliferative layer. Here, the chondroprogenitors generate columns of chondrocytes (5-8 cells deep in mammalian plates; 15-30 deep in avian cartilage). These cells divide in the long axis of the plate so that flattened mother and daughter cells appear to lie on top and slightly to the side of each other. The shape of the cells suggests that they are under axial loading. After a limited number of rounds of proliferation, the cells change their shape and phenotype. These maturing chondrocytes become swollen and express type X collagen as well as type II collagen; in addition, they exhibit high alkaline phosphatase activity. From a functional viewpoint, these hypertrophic chondrocytes secrete membrane-limited vesicles (matrix vesicles) that serve to initiate mineralization of the extracellular matrix. As the cells become hypertrophic, they increase their volume five- to 12-fold and elevate three-fold the total amount of territorial extracellular matrix. When terminally differentiated, the increase in cell volume and the presence of a bulky extracellular matrix provide much of the space required for bone elongation. At the calcified end of the growth plate, trabeculae of woven bone are deposited on the calcified cartilage septa."

"Osteoblasts often occupy chondrocyte lacunae [thus providing some evidence for transdifferentiation]"

"Hypertrophic chondrocytes have been reported to continue to synthesize type I collagen, glycosaminoglycans, and glycolytic and oxidative enzymes and phosphatases"

"terminally differentiated hypertrophic chondrocytes expressed type II collagen, osteopontin, osteocalcin, osteonectin, and aggrecan core-binding protein mRNA."

"when immature sternal chondrocytes were challenged with an apoptogen, they were far more resistant to the induction of apoptosis than were hypertrophic cells"

"treatment of hypertrophic tibial chondrocytes with Pi induced death in a dose- and time-dependent manner. Within 48 hours, 3 mM Pi increased chondrocyte apoptosis by 30%; lower concentrations of Pi induced death after 48 hours. More recently, it was shown that this effect is in large part dependent on another apatitic ion, Ca2+. A modest increase in the Ca2+ concentration from 1.9 to 2.3 mM caused a dramatic increase in chondrocyte death. At a Ca2+ level of 2.8 mM, a small rise in the Pi concentration promoted rapid cell death. Since Ca2+ alone, even at concentrations of 2.8 mM, did not influence the rate of killing, it was concluded that the concentration of the ion pair served as a primary death signal."<-Since hypertophic chondrocytes are so sensative to apoptosis maybe inhibiting this apoptosis can enable these chondrocytes to hypertrophy for longer and enable you to grow taller.

"A null mutation in the MMP-9/gelatinase B gene caused an abnormal pattern of skeletal growth. While terminal differentiation appeared to be normal, apoptosis was delayed, resulting in an eight-fold lengthening of the growth plate."  It's unclear from the study whether this translated into increased adult bone length.

Hyperbaric oxygen treatment prevents nitric oxide-induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70.

"Heat shock proteins (HSPs), inflammatory cytokines, nitric oxide (NO), and localized hypoxia-induced apoptosis are thought to be correlated to the degree of cartilage injury. We investigated the effect of hyperbaric oxygen (HBO) on interleukin-1β (IL-1β)-induced NO production and apoptosis of rabbit chondrocytes and healing of articular cartilage defects. For the in vitro study, RT-PCR and Western blotting were performed to detect mRNA and protein expressions of HSP70, inducible NO synthase (iNOS), and caspase 3 in IL-1β-treated chondrocytes. To clarify that the HSP70 was necessary for anti-iNOS and anti-apoptotic activity by HBO, we treated the cells with an HSP70 inhibitor, KNK437. For the in vivo study, cartilage defects were created in rabbits. The HBO group was exposed to 100% oxygen at 2.5 ATA for 1.5 h a day for 10 weeks. The control group was exposed to normal air. After sacrifice, specimen sections were sent for examination using a scoring system. Immunohistochemical analyses were performed to detect the expressions of iNOS, HSP70, and caspase 3. Our results suggested that HBO upregulated the mRNA and protein expressions of HSP70 and suppressed those of iNOS and caspase 3 in chondrocytes. KNK437 inhibited the HBO-induced downregulation of iNOS and casapase 3 activities. The histological scores showed that HBO markedly enhanced cartilage repair. Immunohistostaining showed that HBO enhanced HSP70 expression and suppressed iNOS and caspase 3 expressions in chondrocytes. Accordingly, HBO treatment prevents NO-induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70."

Apoptosis of growth plate chondrocytes occurs through a mitochondrial pathway.

"Chondrocytes isolated from the growth plates of chick embryo tibia were treated with Pi in serum-free media; chondrocyte viability, mitochondrial membrane potential, cytochrome c release from mitochondria, caspase 3 activity, endonuclease activity, and DNA fragmentation were investigated.

Exposure to Pi for 24 hours induced apoptosis in growth plate chondrocytes through a pathway that involved loss of mitochondrial function, release of cytochrome c into the cytoplasm, increases in caspase 3 and endonuclease activities, and fragmentation of DNA."

"[Apoptotic] cells are characterized by shrinkage of their cytoplasm, condensation of chromatin, blebbing, and formation of vesicles containing the remnants of the cell (apoptotic bodies) that are engulfed by macrophages. During this highly regulated process, there is activation of proteases that lead to cleavage of enzymes and structural proteins and, ultimately, to DNA fragmentation, the hallmark of apoptosis"

"[Alterations occurred in] chondrocytes exposed to 5 mM Pi. Their mitochondria accumulated in the perinuclear region of the healthy cell. Chondrocytes maintained this appearance for the first 4 hours of Pi treatment. Approximately 8 hours after the initiation of treatment, chondrocytes presented signs of stress and began to loose attachment to the glass coverslips; numerous vesicles could be observed in their cytoplasm. While remaining functional, the mitochondria presented a different cellular distribution. After 24 hours of Pi exposure, chondrocytes had shrunk into smaller cellular masses without loosing their membrane integrity. Mitochondria lost their membrane potential. Some of the vesicles in the cytoplasm of these cells contain DNA, the result of endonuclease activity "

"When serum-free media was supplemented with 5 mM Pi, we observed a larger increase in cytochrome c in the cytoplasm fraction. As expected, cytochrome c content was greater in the cell fraction containing mitochondria."

"Release of cytochrome c into the cytosol results in caspase 3 activation and starts a cascade of protease activities (caspases and endonucleases) that ultimately leads to DNA degradation and death. Supplementing serum-free media with 5 mM Pi for 24 hours caused a significant increase in caspase 3 activity in chondrocytes. When the cells were treated with PFA (an inhibitor of Pi cellular transport) before exposure to Pi, caspase 3 activity remained low. "

Growing Taller with Heat

We know that water is a key component of growing taller.  Temperature alters the state of water thus it has the ability to alter hydrostatic pressure.  Something like LIPUS also increases temperature and the speed of chemical reactions.  Studying temperature and the effects of temperature on water and in turn on hydrostatic pressure will help us in our plans for growing taller.

Exercise mitigates the stunting effect of cold temperature on limb elongation in mice by increasing solute delivery to the growth plate.

"Ambient temperature and physical activity modulate bone elongation in mammals. Cartilaginous [growth] plates receive nutritional support via delivery of solutes from the vasculature. We [increased] solute delivery to the growth plate{this doesn't mean that cold and warm temperature modulate hydrostatic pressure too}. We housed 68 weanling female mice at cold (16°C) or warm (25°C) temperatures and allowed some groups voluntary access to a running wheel. Exercise mitigates the stunting effect of cold temperature on limb elongation after 11 days of wheel running. All runners had significantly lengthened limbs, regardless of temperature, while nonrunning mice had shorter limbs that correlated with housing temperature. Tail length was impacted only by temperature, indicating that the exercise effect was localized to limb bones and was not a systemic endocrine reaction{This could be a growth rate only effect however only a small change in temperature was studied}.  [There was] enhanced solute delivery to tibial growth plates in wheel-running mice{Cartilage is negatively charged which helps it attract positive ions which makes it absorb more water so enhanced solute delivery is related to water}, measured under anesthesia at rest. There was a minimal effect of rearing temperature on solute delivery when measured at an intermediate room temperature (20°C), suggesting that a lasting increase in solute delivery is an important factor in exercise-mediated limb lengthening but may not play a role in temperature-mediated limb lengthening{exercise may lead to permanent adaptions in vascularity in the surrounding tissues if not directly in the growth plates in contrast to a temperature change}."

"Growth plate cartilage does not have a penetrating blood supply. Growth plates receive nutritional support via delivery of solutes from the vasculature located in and around the adjacent bone"<-But the efficiency of the surrounding vasculature can impact the delivery of solutes to the growth plate.

"Environmental temperature and exercise may modify extremity growth by inducing changes in limb vasculature"<-Vasculature could also influence hydrostatic pressure too.

"High temperature, exercise, and stimulated muscle contractions increase blood supply to bone" <-This is why flexing may help improve LSJL results.

"Exercise markedly increased fluorescein tracer[fluorescin is a mechanism of measuring solute absorption] intensities in the growth plate and metaphyseal vasculature of wheel-running mice. There was no appreciable effect of temperature on fluorescein levels in the growth plate or vasculature" <-So the increase in vasculature can explain  height grain in the exercise groups but it can't in the temperature groups so likely hydrostatic pressure plays a role there.

"Moderate compression of articular cartilage has an anabolic effect on chondrocytes by stimulating changes in cell matrix, fluid flow, and biochemical activity. Since growth plate cartilage is composed of the same resident cell type, it is possible that increased compression of the long bones stimulates the same changes in growth plate cartilage to enhance its growth potential, whereas temperature acts through alternate mechanisms"<-That mechanism likely being hydrostatic pressure.

"Bone perfusion was only reduced in mice housed at their coldest study temperature (7°C) and did not differ between two warmer-housed groups (21°C and 27°C), even though limb lengths differed. It is possible that the temperatures used in the present study (16°C and 25°C) were above a similar cold threshold that maintains normal solute delivery to cartilage."<-However, the cold temp and warm temperature mice still differed in terms of limb length.

"To increase solute delivery to the growth plate, increase the total volume of blood arriving at a bone by increasing flow rate, enlarging vessel diameter (vasodilation), and/or increasing vessel numbers (angiogenesis)."<-Flow rate being the mechanism likely to enhance hydrostatic pressure.

"The WW[warm wheel] mice had slightly lower levels of fluorescein in the vasculature, yet their relative growth plate levels did not differ from their CW[cold wheel] counterparts."<-So the warm wheel had to be getting their solutes from another source than the vasculature.  However, it could be that cold temperature reduces bone density resulting in less fluorescein per square inch.

So therefore, vascularity explains the increase in bone growth due to exercise but not during temperature changes.  Changes in the composition of water may play a role in temperature differences.  There is very little change in density of water between 0 and 25 degrees celsius so the density of water is not likely to play a role.  The density decreases slightly from 0 to 25 degrees celsuis.  From 0 to 45 degrees celsius the amount of compression water does decreases.  After 45 degrees celsius it starts to rise again.  From cold to warm in the study water would be less compressible.  Less compression of water could lead to higher hydrostatic pressure.

Temperature regulates limb length in homeotherms by directly modulating cartilage growth.

"[A] strong positive correlations exist among latitude, ambient temperature, and limb length in mammals{of course the difference could be due to genotypic selection rather than adaptions in phenotype}. Although genetic selection for thermoregulatory adaptation is frequently presumed to be the primary basis of this phenomenon, important but frequently overlooked research has shown that appendage outgrowth is also markedly influenced by environmental temperature{but is this influence only in growth rate or does it affect adult stature}. Alteration of limb blood flow via vasoconstriction/vasodilation [partially explains] this growth plasticity{Water is likely involved and we can see what properties of water are involved by seeing what happens to the growth at which temperatures which correlate with different properties of water}. Peripheral tissue temperature closely reflects housing temperature in vivo, and chondrocyte proliferation and extracellular matrix volume strongly correlate with tissue temperature in metatarsals cultured without vasculature in vitro{Chondrocyte proliferation increase can still be inhibited by a limit in proliferative capacity so temperature increase wouldn't affect adult stature.  ECM volume could impact adult stature but does ECM volume always increase with increasing temperature}. Vasomotor changes likely modulate extremity growth indirectly, via their effects on appendage temperature, rather than vascular nutrient delivery{appendage temperature affecting the properties of the compounds inside the appendeges like water}."

"Impairment of bone growth by disruption of blood flow is well-known, as are correlations of enhanced blood flow with bone elongation and ear enlargement"

"The ears, limbs, and tails of warm-reared mice were significantly longer than those of siblings raised in the cold, with no change in total body mass"<-If cellular proliferation was impacted then you'd think there would be an increase in cellular proliferation everywhere.

"Differences in core organ size appeared to account for the latter—hearts and kidneys were enlarged in cold-reared mice—{you'd think you'd need more than enlarged heart and kidneys to compensate for limb length gain} but differences in limb length were not explicable by diet and/or activity level, because cold-reared mice consumed substantially more food and were more active than their warm and control counterparts"

"Blood not only provides developing limbs with a reservoir of essential growth factors, nutrients, and oxygen, it is also an important source of heat"<-The larger the blood vessals the more heat available for the chondrocytes for chemical reactions.

"growth of neonatal mouse metatarsals (MTs) in culture maintained at cold (32 °C), control (37 °C) or warm (39 °C) temperatures"

"MTs from all three groups showed increases in length from baseline at two- and four-day time intervals, but total accrued growth was strikingly temperature dependent. Interestingly, although the control and warm groups differed only slightly in temperature (2 °C), this small increase was still sufficient to produce significantly greater growth in warm MTs compared with both other groups"

So temperature seems to be beneficial at least up to 39 degrees Celsius.  Room Temperature is usually between 15 to 25 degrees celsius.  Saunas are typically between 70 and 100 degrees celsius.  A hot tub can go to 99 or 104.  If the growth benefits are related to water and compressibility you'd expect to see a decline in benefits around 45 degrees celsius.  It's also unclear whether the benefits of heat need to be sustained or can they be intermittent.  For example, a heating pad or the hot tub/jacuzzi.   Increased muscle mass could lead to elevated temperatures.

Increased blood vessel size and larger ECM could lead to higher growth during puberty as well as during LSJL.  As the temperature could impact the ability of stem cells to differentiate into chondrocytes.

Hindlimb heating increases vascular access of large molecules to murine tibial growth plates measured by in vivo multiphoton imaging.

"Dense extracellular matrix and lack of penetrating blood vessels create a semi-permeable "barrier," which hinders molecular transport at the vascular-cartilage interface. To overcome this obstacle, we employed a hindlimb heating model to manipulate bone circulation in 5-week-old female mice. Temperatures represented a physiological range of normal human knee joints. We used in vivo multiphoton microscopy to quantify temperature-enhanced delivery of large molecules into tibial growth plates. We tested the hypothesis that increasing hindlimb temperature from 22C to 34C increases vascular access of large systemic molecules, modeled using 10-, 40-, and 70 kDa dextrans that approximate sizes of physiological regulators. Vascular access was quantified by vessel diameter, velocity, and dextran leakage from subperichondrial plexus vessels and accumulation in growth plate cartilage. Growth plate entry of 10 kDa dextrans increased over 150% at 34C. Entry of 40- and 70 kDa dextrans increased < 50%, suggesting a size-dependent temperature enhancement. Total dextran levels in the plexus increased at 34C, but relative leakage out of vessels was not temperature-dependent. Blood velocity and vessel diameter increased 118% and 31%, respectively, at 34C. These results demonstrate that heat enhances vascular carrying capacity and bioavailability of large molecules around growth plates, suggesting that temperature could be a non-invasive strategy for modulating delivery of therapeutics to impaired growth plates of children."

But can this be used to increase height?

"There are two components of the vascular-cartilage barrier that determine solute availability in growth plates: 1) ability of molecules to exit the vasculature and 2) ability to enter the cartilage matrix. The endpoint of interest is total uptake in the growth plate since this is what ultimately impacts chondrocyte performance. Molecular size [is] a limiting factor for entry of fluoresceinated dextrans into mouse tibial growth plates.  A transport block [exists] at the metaphyseal chondro-osseous junction and molecules over 10 kDa [are] essentially size-excluded from the growth plate."

"heat increases 107 small solute (< 500 Da) uptake in growth plate cartilage in vivo"

One question is where there is any large molecules produced by the human body naturally or are available in foods and supplements that affect height?

Weightlifting may increase height?

Thickening of the knee joint cartilage in elite weightlifters as a potential adaptation mechanism.

"Thickening and increase of area of cartilage have been proposed as two alternative mechanisms of cartilage functional adaptation. The latter has been reported in endurance sportsmen. In weightlifters, extreme strain applied to the articular surfaces can result in other forms of adaptation. The aim of this research is to determine whether cartilage thickness is greater in elite weightlifters than in physically inactive men. Weightlifters (13) and 20 controls [age and body mass index (BMI) matched] underwent knee Magnetic Resonance Imaging (MRI). A single sagittal slice of the knee was taken and cartilage thickness was measured in five and six regions of the medial and lateral femoral condyles, respectively. The analyzed segments represented weight-bearing and nonweight-bearing regions. The tibia cartilage in the weight-bearing area was also measured. The time of training onset and its duration in the weightlifter group were recorded. The cartilage was found to be significantly thicker in weightlifters in most of the analyzed regions. The distribution of cartilage thickness on the medial and lateral femoral condyles was similar in both groups. The duration of training was not associated with cartilage thickness, but the time of training onset correlated inversely with cartilage thickness. It is possible that in high-strain sports, joint cartilage can undergo functional adaptation by thickening. Thus, mechanical loading history could exert a postnatal influence on cartilage morphology."

The Weightlifters were members of the Polish weightlifting team.

"Method of selection measurement points for cartilage thickness evaluation on medial (A and B) and lateral (C and D) femoral condyles. Cartilage thickness was measured perpendicularly from the point of crossing of the subchondral bone surface with the line determining particular angle."

"The weightlifters had a mean age of 26.1 years and a mean BMI of 29.1. They did not differ significantly from the control group. The mean cartilage thickness in all analyzed regions of both condyles was greater in weightlifters than in the controls(athletes vs. control—lateral condyle: 2.9, SD = 1.1 vs. 2.1, SD = 0.9, P = 0.0000; medial condyle: 2.9, SD = 0.9 vs. 2.1, SD = 0.8, P = 0.0000)."

The lowest age tested was 19 years old.  The average height height of the weightlifters was lower than control.

"knee cartilage morphology of seven elite weightlifters with men who had never performed strength training and observed that cartilage thickness was not significantly greater in the group of athletes, except for a 14% thicker patella cartilage. Those results are even more surprising because all athletes had been actively training throughout adolescence, and they displayed 30% higher extensor mean cross sectional areas than the nonathletic volunteers. "

"ecrease in cartilage thickness of 30 ± 10% in the femoral trochlea after 3.5 h of static loading with 150% body weight. However, only about 4–7% of the final deformation is reached during the first minute of loading."

It seems that cartilage thickness does not affect height.

Grow Taller by Increasing Chromatin Acetylation?

Recently, we discovered that chromatin hyperacetylation was critical in inducing SOX9 transcription activity.  Stimulation of histone acetyltransferase or inhibition of histone deactylase may be a way to grow taller.  Low levels of chromatin acetylation results in less of the ECM genes aggrecan and COL2A1 which may interfere with optimal growth.

Chromatin hyperacetylation is an epigentic mechanism.  Epigenetics can be transferred between generations.  Transcription states are heritable.  The status of the parent at that particular moment may affect a child's development.  Other examples of epigentics are telomere length and DNA Methylation Status.

How does chromatin acetylation on SOX9 affect height and how do we increase chromatin acetylation?

Histone acetylation influences the activity of Sox9-related transcriptional complex.

"From the mesenchymal condensation of chondroprogenitors to the hypertrophic maturation of chondrocytes, chondrogenesis is sequentially regulated by cross-talk among transcription factors, growth factors, and chromatin structure{altering chromatin structure can alter chondrogenesis}. The master transcription factor Sry-type HMG box (Sox) 9 has an essential role in the expression of chondrogenic genes through the association with Sox9-binding sites on its target genes{thus to grow taller it's essential that Sox9 is working properl}. Several transcription factors and coactivators, such as Scleraxis/E47 and p300, cooperatively modulate the Sox9-dependent transcription by interacting with Sox9. The Sox9-related transcriptional apparatus activates its target gene expression through p300-mediated histone acetylation on chromatin. The transforming growth factor (TGF)-β superfamily also plays a key role in chondrocyte differentiation. The TGF-β-regulated Smad3/4 complex activates Sox9-dependent transcription on chromatin by associating with Sox9 itself, and by recruiting p300 onto Sox9[thus TGF-Beta may help with Sox9 transcription, note LSJL increases TGF-Beta levels]. The epigenetic status including histone modification and chromatin structure, directly influences Sox9-regulated chondrocyte differentiation."

The degree of chromatin folding affects transcriptional activity and less folding is better for activating transcription activity.

"Protein kinase A-induced phosphorylation of Sox9 enhances Sox9-dependent transcription by increasing the DNA-binding affinity of Sox9. On the other hand, Sox9 activity is suppressed by PIAS1-mediated sumoylation of Sox9. The ubiquitin-proteasome pathway also inhibits Sox9 transcriptional activity by inducing the degradation of Sox9"

"In the absence of Sox5/6, sclerotome MSCs are prevented from differentiating into chondrocytes, and switch their fate to Scx-expressing tendon/ligament progenitors"

"The MYST family coactivator Tip60, which mainly acetylates H4, increases Sox9/Sox5-dependent Col2a1 transcription by associating with Sox9 on chromatin"

"Sox5/6 may stabilize Sox9 on its binding site through the bending of DNA and thereby stimulate Sox9-regulated gene expression"

"Fibroblast growth factor (FGF) 1, FGF2, and insulin-like growth factor 1 up-regulate the expression of Sox9"

"Histone deacetylase (HDAC) inhibitors, including trichostatin A (TSA) and FK228, have the synergistic potential to induce Sox9 expression via enhanced recruitment of nuclear factor Y (NF-Y) to the proximal promoter of Sox9"

"IL-1βtreatment down-regulates Sox9 transactivation by a reduction of Sp1 binding to the Sox9 promoter"

BMP-2 induces histone hyperacetylation on Chromatin in Sox9.  This could be why too much BMP-2 reduces height growth.

"the BMP-2 inhibitor Noggin represses Sox9 expression in limb bud chondrogenic precursors while
inducing the ligament/tendon-specific transcription factor Scx"

"the histone acetyltransferase (HAT) activity of p300 has the potential to facilitate transcriptional activity by modulating the chromatin structure. In chondrogenesis, p300 stimulates transcription factor-mediated chromatin disruption. The CH3 domain of coactivator p300 directly associates with the C-terminal PQ-rich transactivation domain of Sox9, and activates Sox9-dependent transcription in chondrogenesis"

"Smad3 also stabilized the association between Sox9 and p300 by forming a transcriptional apparatus with Sox9 and p300"

"the bHLH transcription factor Scx{up in LSJL} and its partner E47 cooperatively stimulated Sox9-dependent transcription through the formation of a transcriptional complex with Sox9 and p300"

"Sox9 interacts with the Med12/Trap230 subunit of the mediator complex to stimulate RNA polymerase II-dependent transcription in chondrocytes. Med12/Trap230 acts as an essential bridging factor between Sox9 and the RNA polymerase II transcriptional machinery. Peroxisome proliferator activated receptorγcoactivator 1α(Pgc1α), which is involved in gluconeogenesis, stimulates Sox9- dependent transcription including Col2a1 and COMP expression via direct association with Sox9. Sox9 and the homeobox transcription factor Barx2{up} cooperatively bind to adjacent sites in the Col2a1 enhancer, and regulate chondrogenesis during limb development"

"histone hyperacetylation using the HDAC inhibitor TSA enhanced Sox9-regulated cartilage matrix gene expressions (COL2A1 and aggrecan) in human chondrocytes"

Being transgenic in Sox9 results in dwarfism.

Interactions between Sox9 and beta-catenin control chondrocyte differentiation.

"Here we show the existence of physical and functional interactions between beta-catenin and Sox9, a transcription factor that is required in successive steps of chondrogenesis. In vivo, either overexpression of Sox9 or inactivation of beta-catenin in chondrocytes of mouse embryos produces a similar phenotype of dwarfism with decreased chondrocyte proliferation, delayed hypertrophic chondrocyte differentiation, and endochondral bone formation[thus the best way to optimize Sox9 would be to find the equilibrium quantity of BMP-2]. Furthermore, either inactivation of Sox9 or stabilization of beta-catenin in chondrocytes also produces a similar phenotype of severe chondrodysplasia. Sox9 markedly inhibits activation of beta-catenin-dependent promoters and stimulates degradation of beta-catenin by the ubiquitination/proteasome pathway[too much Sox9 inhibits activation of Beta-Catenin so a way to counteract the dwarfism caused by too much Sox9 you'd want to stimulate more production of Beta-Catenin]. Likewise, Sox9 inhibits beta-catenin-mediated secondary axis induction in Xenopus embryos. Beta-catenin physically interacts through its Armadillo repeats with the C-terminal transactivation domain of Sox9. We hypothesize that the inhibitory activity of Sox9 is caused by its ability to compete with Tcf/Lef for binding to beta-catenin, followed by degradation of beta-catenin. Our results strongly suggest that chondrogenesis is controlled by interactions between Sox9 and the Wnt/beta-catenin signaling pathway."

"In the canonical Wnt pathway, binding of secreted Wnts to the Frizzled family of cell surface receptors inactivates Gsk3-β, resulting in stabilization and nuclear translocation of β-catenin and activation of Wnt target genes. The noncanonical pathways also signal through the Frizzled receptors. The planar cell polarity pathway activates the rho family of GTPases and the Jun N-terminal kinase, and modifies cytoskeletal organization and epithelial cell polarization. The Wnt/Ca²⁺ pathway stimulates the intracellular increase of Ca²⁺ through activation of protein kinase C and calmodulin-dependent kinase II."<-Lithium does this too.
 
"In vitro studies of chondrogenic mesenchymal cells in micromass cultures show that LiCl, which inhibits Gsk3-β and mimics canonical Wnt pathway activation, or a proteasome inhibitor, which stabilizes β-catenin, as well as overexpression of stβ-catenin, markedly inhibit expression of the major chondrocyte marker Col2a1, suggesting that β-catenin inhibits overt chondrocyte differentiation"<-Thus increasing Lithium levels may be a way to compensate for increasing Sox9 levels.

"Expression of the Cyclin D1 gene and the activity of its promoter are increased by the β-catenin/Tcf-Lef complex. Sox9 overexpression in chondrocytes in vivo induces a marked down-regulation of Cyclin D1. Sox9 also inhibits the β-catenin-mediated increase in Cyclin D1 promoter activity"<-It may be best to just find a way to directly stimulate Cyclin D1 expression.

"Overexpression of Sox9 in committed chondrogenic mesenchymal cells did not affect cell viability (data not shown) as well as the timing and the overall size of mesenchymal condensations, indicating that although Sox9 is required for chondrogenic mesenchymal condensations, a small amount of additional Sox9 has no effect on chondroprogenitors in vivo. Moreover, in Sox9-overexpressing embryos, overt differentiation into chondrocytes of cells present in mesenchymal condensations occurred normally."

"Cyclin D1 controls progression through the G1 phase of the cell cycle, and its expression is strictly regulated by transcriptional mechanisms. In growth plates, Cyclin D1 is specifically expressed in proliferating chondrocytes"<-thus being transgenic in Cyclin D1 may enable more chondrocyte proliferation.

"Sox9 also controls chondrocyte proliferation by inhibition of Cyclin D1 expression."<-Sox9 is required for mesenchymal differentiation into chondrocytes but it also inhibits chondrocyte proliferation by inhibiting Cyclin D1.

"This small additional amount of Sox9 is sufficient to produce a very abnormal skeletal phenotype."<-thus it is crucial not to increase chromatin acetylation unless you're deficient.

"Expression of the Cyclin D1 gene and the activity of its promoter are increased by the β-catenin/Tcf-Lef complex."

Here's a study on the HMGA2 gene which is related to chromatin activation and affects height:

HMGA2, MicroRNAs, and Stem Cell Aging

"a chromatin-associated protein, HMGA2, [is linked to] development, height, and mouse stem cell aging during late fetal development and young adulthood"<-Thus Chromatin may be linked to height as well.

"identifies the chromatin-associated protein HMGA2 as a developmental regulator of stem cell self-renewal"

"an association between common alleles of SNPs linked to HMGA2 and human height"

"A spontaneous mutation of the Hmga2 gene has been previously shown to result in the murine pygmy phenotype, which includes reduced adult size. Meanwhile, transgenic mice that overexpress a wild-type Hmga2 gene or a truncated variant without the 3′ UTR exhibit gigantism. This latter Hmga2 allele is of interest because the 3′ UTR truncation removes the let-7 binding sites, thereby abrogating the repression of Hmga2 by let-7. Furthermore, a germline chromosomal inversion that results in a similarly truncated human HMGA2 gene was identified in a boy with severe overgrowth. The HMGA2-linked SNPs that are most strongly associated with height are known to lie within the 3′ UTR of HMGA2, suggesting that the height-influencing genetic events linked to these SNPs may influence let-7 binding"<-Thus we should analyze let-7 and it's effect on height growth.

Conclusion:  Increasing chromatin acetylation may be a way to grow taller if you are deficient.  The best way to do so is via BMP-2 but genetic expression of Sox9 and Beta-Catenin must be carefully monitored.  Increasing Beta-Catenin levels may be possible with Lithium which stabilizes Beta-Catenin.  Instances of too high levels of Beta-Catenin have not been found to cause dwarfism however.  Although, there is likely a limited number of Beta-Catenin to stabilize.  However, increasing Sox9 activation may be viable as long as there is more Beta-Catenin activation in the system to ensure proper functioning of the Cyclin D1 gene to ensure proper chondrocyte proliferation.

Transcriptional regulation of chondrogenesis by coactivator Tip60 via chromatin association with Sox9 and Sox5.

"Here, we report on the characterization of a Tat interactive protein-60 (Tip60) as Sox9-associated protein identified in a yeast two-hybrid screen. Both in vitro and in vivo assays confirmed the specificity of interactions between Sox9 and Tip60 including the existence of an endogenous complex containing both polypeptides in chondrocytes. Gel shift assays showed the presence of a complex containing Sox9, Tip60 and the DNA of an enhancer region of the Col2a1 promoter. Reporter assays using a Col2a1 promoter with multimerized Col2a1 Sox9-binding sites indicated that Tip60 enhanced the transcriptional activity of Sox9. A larger Col2a1 promoter showed that Tip60 increased the activity of this promoter in the presence of both Sox9 and Sox5. Ectopic expression of Sox9 and transient-cotransfection with Tip60 in COS7 cells showed a more diffuse subnuclear colocalization, suggesting changes in the chromatin structure. Chromatin immunoprecipitation assays showed that Tip60, Sox9 and Sox5 associated with the same Col2a1 enhancer region. Consistent with a role of Tip60 in chondrogenesis, addition of Tip60 siRNA to limb-bud micromass cultures delayed chondrocyte differention. Tip60 enhances acetylation of Sox9 mainly through K61, 253, 398 residues; however, the K61/253/398A mutant of Sox9 still exhibited enhanced transcriptional activity by Tip60. Our results support the hypothesis that Tip60 is a coactivator of Sox9 in chondrocytes."

"Posttranslational modifications of nucleosomal histones have been proposed to influence chromatin structure and to create a code that is interpreted by positive and negative transcriptional regulators recognizing specific histone modifications. Histone acetylation, catalyzed by histone acetyl transferase (HAT), promotes gene transcription by relaxing the chromatin structure, thereby facilitating access of the transcriptional machinery to DNA target sequences. The transcription-activating effect of histone acetylation is counterbalanced by histone deacetylation, which favors chromatin condensation and transcriptional repression"

"[Tip60] acetylates histone H4, modulates DNA-damage response signaling, which is triggered by oncogenes, and controls cell cycle checkpoints and apoptosis"

"Tip60 is a tightly regulated transcriptional coactivator for androgen-, estrogen- and progesterone- receptors. It also acts as a coactivator of p53 in activation of the p21 promoter and has been found on c-Myc and NFKB target genes"

HISTONE DEACETYLATION REGULATES GROWTH PLATE DEVELOPMENT

"Histone deacetylation 4 (HDAC4) nuclear-cytoplasmic shuttling, degradation, and translational repression plays a major role in [the growth plate]. Specifically, we suspect that HDAC4 functions as a negative regulator of chondrocyte hypertrophy by binding and inhibiting Runx 2/Cbfa1 expression in the nucleus. Runx 2/Cbfa1 is a transcription factor necessary for chondrocyte differentiation and hypertrophy.

Our overall hypothesis is that the relocation, degradation, and translational repression of HDAC4 control chondrocyte differentiation and are regulated by Ca2+/calmodulin kinase IV (CaMKIV), p38 MAP kinase, and microRNA-1 respectively at different zones in the growth plate.

Specific Aim 1: To determine whether activation of the Ca2+/calmodulin signaling pathway prevents nuclear entry of HDAC4 and enhances the binding of HDAC4 to the cytoplasmic binding protein 14-3-3. This may impair HDAC4-mediated inhibition of chondrocyte differentiation in the nucleus[so by stimulating calmodlulin you could stimulate chondrogenesis]. Hypothesis 1 is that HDAC4 nuclear- cytoplasmic shuttling controls chondrocyte differentiation and is dependent on the Ca2+/calmodulin signaling pathway.
Specific Aim 2: To determine whether: 2a) caspases degrade HDAC4; 2b) p38 MAPK regulates expression or activity of capsases 2 and 3; 2c) Runx2 expression is dependent on p38 MAPK by in situ hybridization in p38 MAPK defective and constitutively activated MKK6 mouse growth plates. Project 2b will be tested using active MKK6 to elevate p38 and dominant negative p38 MAPK to repress p38. Hypothesis 2 is that HDAC4 degradation is regulated by P38 MAPK by increasing expression of caspases 2 and 3.
Specific Aim 3: To determine whether: 3a) the spatio-temporal distribution of specific miRNA-1 is different during growth plate development; 3b) miRNA-1 downregulates HDAC4 by repressing HDAC4 translation at 3' UTR in the chondrocytes; 3c) miRNA-1regulates chondrocyte proliferation and differentiation.
Hypothesis 3 is that microRNA-1 is involved in chondrocyte hypertrophy regulation by repressing HDAC4 translation. Delineating the physiological controls of the physis could suggest possibilities for therapeutically manipulating endochondral bone growth by modulating the signaling pathways that govern the association of HDAC4 with Runx2. Prevention of leg length discrepancy, dwarfism, and other disorders of bone growth might also be possible once molecular triggers and stops affecting the growth plate are better understood."

Exposure to Valproic Acid Inhibits Chondrogenesis and Osteogenesis in Mid-Organogenesis Mouse Limbs.

"In utero exposure to valproic acid (VPA), a histone deacetylase (HDAC){so this tells us the important of histone deacetylation on height growth} inhibitor, causes neural tube, heart and limb defects. Valpromide (VPD), the amide derivative of VPA, does not inhibit HDAC activity and is a weak teratogen in vivo. The detailed mechanism of action of VPA as a teratogen is not known. The goal of these studies was to test the hypothesis that VPA disrupts regulation of the expression of genes that are critical in chondrogenesis and osteogenesis during limb development. Murine gestation day 12 embryonic forelimbs were excised and exposed to VPA or VPD in a limb bud culture system. VPA caused a significant concentration-dependent increase in limb abnormalities that was correlated with its HDAC inhibitory effect. The signaling of both Sox9 and Runx2, key regulators of chondrogenesis, was downregulated by VPA. In contrast, VPD had little effect on limb morphology and no significant effect on HDAC activity or the expression of marker genes. Thus, VPA exposure dysregulated the expression of target genes directly involved in chondrogenesis and osteogenesis in the developing limb. Disturbances in these signaling pathways are likely to be a consequence of HDAC inhibition since VPD did not affect their expression."

"VPA is an inhibitor of class I and II histone deacetylases (HDACs)"

"apicidin, MS-275, sodium butyrate and sodium salicylate{Sodium salicylate is present in some treatments, you may want to avoid it} are all HDAC inhibitors that induce defects of the axial skeleton in mice"

"the low dose VPA treatment group showed minimal effects on morphology, the limbs exposed to 1.8 or 3.6 mM VPA exhibited a marked decrease in growth and differentiation. The long bones were reduced in size, the staining of the carpalia was decreased and the metacarpals were short and thick."

"VPA exposure resulted in the downregulation of Sox9 mRNA expression by 1h in all VPA-exposed limbs"

The high mobility group protein HMGA2: a co-regulator of chromatin structure and pluripotency in stem cells?

"[HMGA2] proteins are abundant in pluripotent embryonic stem (ES) cells and most malignant human tumors, but are not detectable in normal somatic cells. They act both as activator and repressor of gene expression, and most likely facilitate DNA architectural changes during formation of specialized nucleoprotein structures at selected promoter regions. For example, HMGA2 is involved in transcriptional activation of certain cell proliferation genes, which likely contributes to its well-established oncogenic potential during tumor formation. However, surprisingly little is known about how HMGA proteins bind DNA packaged in chromatin and how this affects the chromatin structure at a larger scale. Experimental evidence suggests that HMGA2 competes with binding of histone H1 in the chromatin fiber. This could substantially alter chromatin domain structures in ES cells and contribute to the activation of certain transcription networks. HMGA2 also seems capable of recruiting enzymes directly involved in histone modifications to trigger gene expression. Furthermore, it was shown that multiple HMGA2 molecules bind stably to a single nucleosome core particle whose structure is known. How these features of HMGA2 impinge on chromatin organization inside a living cell is unknown. In this commentary, we propose that HMGA2, through the action of three independent DNA binding domains, substantially contributes to the plasticity of ES cell chromatin and is involved in the maintenance of a un-differentiated cell state."

"HMGA2 [controls] (stem) cell proliferation and the development of adipose tissue."

"During activation of E2F1{down in LSJL}, HMGA2 replaces the histone deacetylase from DNA and recruits histone acetylase, thus leading to sustained gene expression from this locus"  E2F1 binds to Sp1 which binds to Sox9(Source: String DB).

"ERCC1 [is repressed] through binding of HMGA2 to a particular AT-rich site within the promoter region" ERCC1 is several steps removed from Sox9(Source: String DB).

LSJL Update-Bone Length Increase

Here's the link to the last set of pictures.  Here's the latest pictures:

The ruler is over so I can make sure to get the right angle measurements.  Last Measurements:  We had the epiphysis at around 3 7/8".  The increments in the tape measurer are blurry so I've labeled some key points.  The finger experiment is going slowly.  The epiphysis now looks to be about 4 1/8".  In these pictures growth was put into question as the 13 1/4" was the distance achieved in both the before and after pictures.  However, in the before picture the ruler is at a diagonal.  The shortest distance between two points is a straight line therefore the before picture must be shorter than 13 1/4" indicating some growth.  There does seem to be any growth in the diaphysis of the bone in comparing the photos here and the after photos.  For instance, the maximum of the calf comes at about 8 and slopes downward to about 17 1/2.  In the pictures linked there the maximum of calf sarts at about 7 and continues sloping down until about 16.  Leading to a possible half an inch growth there.

It's not absolute proof of anything except that my measurement techniques need work.  But I'm working hard both on performing LSJL and learning the science behind it.  And on performing better measurements.