Two Plants to help growth pre-senescence

Effect of combined extract of Hansogdan (Phlomis umbrosa) and Dalgaebi (Commelina communis) as a milk additive for enhancing the growth of physical height in vivo

"Three different dosage groups for the mixed plant extract of hansogdan (Phlomis umbrosa) and dalgaebi (Commelina communis) (5, 25, and 100 mg/kg/day respectively) were evaluated in rats for the investigation of the enhancement of growth of physical height as a milk additive. Comparing to control group, the nose-to-tail (N-T) length was significantly increased in all treatment groups with a dose dependent manner, nose-to-anus (N-A) length was increased significantly in 25 and 100 mg/kg groups, the increase in both N-T and N-A lengths in 100 mg/kg group was almost same to that of positive group, and the femoral bone length in 25 mg/kg group was significantly increased. The consumption of the mixed extract with milk could stimulate the longitudinal growth and that the addition of the mixed herbal extract into the milk could be more effective than milk itself in terms of the growth of physical height in rats."

Need this full study.

Calgranulin

Calgranulin is used to remove kidney stones which can occur via excessive calcificationn in the kidney.  Calgranulin may be a way to deossify the bone allowing for new growth plate formation.  S100A8/S100A9/S100A12 genes are all associated with calgranulin.


S100A8/S100A9 and their association with cartilage and bone.

"S100A8 and S100A9 are calcium-binding proteins expressed in myeloid cells and are markers of numerous inflammatory diseases in humans. S100A9 has been associated with dystrophic calcification in human atherosclerosis. S100A8 and S100A9 [are expressed] in murine and human bone and cartilage cells. Only S100A8 was seen in preosteogenic cells whereas osteoblasts had variable, but generally weak expression of both proteins.  S100A8 and S100A9 were prominent in osteoclasts. S100A8 was expressed in alkaline phosphatase-positive hypertrophic chondrocytes, but not in proliferating chondrocytes within the growth plate where the cartilaginous matrix was calcifying. S100A9 was only evident in the invading vascular osteogenic tissue penetrating the degenerating chondrocytic zone adjacent to the primary spongiosa, where S100A8 was also expressed. Whilst, S100A8 has been shown to be associated with osteoblast differentiation, both S100A8 and S100A9 may contribute to calcification of the cartilage matrix and its replacement with trabecular bone, and to regulation of redox in bone resorption."

"S100A4{up in LSJL} was found in differentiating osteoblasts and was shown to diminish bone matrix mineralization. S100A2 was detected in chondrocytes within calcifying areas of epiphyseal cartilage suggesting its involvement in cartilage calcification"

"low concentrations of H2O2 or O2− accelerate resorption by osteoclasts and HOCl and hydroxyl radicals can degrade proteoglycans and fragment collagen. HOCl can also alter matrix metalloproteinase (MMP) activity by inactivating tissue inhibitors of MMP and activating certain MMPs "

According to Induction of nuclear factor-κB responses by the S100A9 protein is Toll-like receptor-4-dependent., S100A9 activates NFkappaB via TLR4.

There are several osteoarthritis studies that suggests that S100A8/S100A9 degrade cartilage as well as bone.

Microarray profile of gene expression during osteoclast differentiation in modelled microgravity.

"Microgravity (µXg) leads to a 10-15% loss of bone mass in astronauts during space flight. Osteoclast (OCL) is the multinucleated bone-resorbing cell. In this study, we used the NASA developed ground-based rotating wall vessel bioreactor (RWV), rotary cell culture system (RCCS) to simulate µXg conditions and demonstrated a significant increase (2-fold) in osteoclastogenesis compared to normal gravity control (Xg). Gene expression profiling of RAW 264.7 OCL progenitor cells in modelled µXg by Agilent microarray analysis revealed significantly increased expression of critical molecules such as cytokines/growth factors, proteases and signalling proteins, which play an important role in enhanced OCL differentiation/function. Transcription factors such as c-Jun{up}, MITF and CREB implicated in OCL differentiation are upregulated; however no significant change in the levels of NFATc1 expression in preosteoclast cells subjected to modelled µXg. We also identified high-level expression of calcium-binding protein, S100A8 (calcium-binding protein molecule A8/calgranulin A) in preosteoclast cells under µXg. Furthermore, modelled µXg stimulated RAW 264.7 cells showed elevated cytosolic calcium (Ca(2+)) levels/oscillations compared to Xg cells. siRNA knock-down of S100A8 expression in RAW 264.7 cells resulted in a significant decrease in modelled µXg stimulated OCL differentiation. We also identified elevated levels of phospho-CREB in preosteoclast cells subjected to modelled µXg compared to Xg."

"In space, astronauts are exposed to µXg; 0.08–0.008g rather than Earths inherent gravity of 1g. "

"isolated fetal mouse long bones under near weightless conditions showed decreased mineralization and increased calcium release"

" µXg has been reported to reduce osteoblast life-span and enhance IL-6{up} gene expression, thereby decreasing osteoblast activity and increasing OCL activity to contribute to bone loss associated with weightlessness"

Genes upregulated by microgravity in osteoclasts also upregulated in LSJL:

Pdpn

IL33

CCL24

Edil3

Ppfibp2{down}
Anxa3{down}
Socs3
Pbxip{down}
Vav3{down}
Creb3l1
Mall
Jam2
Masp1
Itpr3{down}
Cacna2d2{down}
Abca9{down}

Down:
Estrogen Receptor Beta(as ESR2){up}

p58 repressor(as Prkrir)

Neurons

Neuronal CaSRs regulate general growth, skeletal development, and energy homeostasis

"To assess biological functions of neuronal CaSR in vivo we generated mice with CaSR knockout (KO) targeted to neurons. The KO mice were viable but growth-retarded with smaller undermineralized skeleton. These growth and skeletal defects were associated with dysregulation of hypothalamus (Hyp)–pituitary gland (Pit)–GH/IGF1 axis in the KO mice, reflected by decreased serum GH and IGF1 levels and reduced GH and GHRH RNA levels in their Pit and Hyp, respectively. Genomic analyses confirmed that CaSR gene excision didn't occur in liver, cartilage, and bones and Western blotting showed intact CaSR expression in the Pit of the KO mice, supporting the primary effects of CaSR KO in Hyp neurons on the growth defects. In the KO mice, the expression of TSH, ACTH, FSH, and LH RNA in their Pit and expression of TRH and GnRH in their Hyp were also decreased by 35–60%. These data together indicate that CaSR KO caused general dysregulation of Hyp functions and produced a panhypopituitarism in the KO mice. Furthermore, in the Hyp of KO mice, expressions of agouti-related protein and neuropeptide Y, which enhance feeding and decrease energy expenditure, were increased by 70 and 25%, respectively, while pro-opiomelanocortin, which suppresses feeding and increases energy expenditure, was decreased by ≈ 30%. These changes in gene expression were accompanied by increased body fat and glucose-intolerance despite their elevated serum leptin levels, suggesting increased leptin-resistance in the KO mice. Together, our data support a critical role for neuronal CaSRs in the integration of general growth, skeletal development, and energy homeostasis."

Osterix

Differential gene expression by Osterix knockdown in mouse chondrogenic ATDC5 cells.

ATDC5 cells are more look chondrocyte progenitor cells than stem cells.

"Osterix (Osx) is a transcription factor required for osteoblast differentiation during intramembranous and endochondral ossification. In an vitro study, which the effects of Osx gene silencing were examined in mouse chondrogenic ATDC5 cells, chondrocyte marker genes were found to be expressionally downregulated and chondrocyte differentiation to be reduced. On the other hand, in vivo studies based on chondrocyte-specific Osx knockouts demonstrated impaired endochondral bone formation with delayed chondrocyte differentiation and reduced cartilage matrix ossification. We investigate global gene expression profile changes caused by Osx knockdown in ATDC5 chondrocytes. The mRNA expressions of 112 genes were significantly modified by Osx knockdown: 68 genes were upregulated and 44 genes downregulated. Functional categories of gene expression classified by gene ontology demonstrated that genes related to cell adhesion, development, and signal transduction were highly affected by Osx knockdown. The expressions of differential genes, such as Sfrp2, Sema3a, Nox4, Rgs4, Zfp521, Has2, Sox6, Scn2a1, Sirpa, and Thbs2, were validated by quantitative real-time PCR."

"Osx gene silencing in mouse chondrogenic ATDC5 cells caused the downregulated expression of chondrocyte marker genes and reduced chondrocyte differentiation"

"Rgs5, Rgs7, and Rgs10 promote chondrocyte differentiation, Rgs4 has an inhibitory effect."

"In chondrocytes, Zfp521 acts on a downstream target gene of PTHR1 signaling and regulates cellular proliferation and differentiation"

"chondrocyte-specific Has2 inactivation causes skeletal deformities and abnormal organization within chondrocytes"

Genes upregulated in Osx knockdown ATDC5 cells also up in LSJL:

Thbs2

Serinc2

Cadm1{down}

Pcsk2{down}

Plscr2{down}

Ppp1r3c

Embigin{down as A430106F12Rik}

Aspn

Htra1

Fgd4{down}

Downregulated:

Anxa8{up}

Sdpr

Gpr115{up}

Creb3l1{up}

Cdh18{up}

Rcbtb2

Rbm26

Haploinsufficiency of Osterix in Chondrocytes Impairs Skeletal Growth in Mice.

"Osteoblast-specific ablation of Osx using Col1α1-Cre resulted in osteopenia, due to impaired osteoblast differentiation in adult mice. Osx is expressed in chondrocytes. We [examine] the skeletal phenotype of mice with conditional disruption of Osx in Col2α1 expressing chondrocytes. Surprisingly, Cre-positive mice that were homozygous for Osx floxed alleles died after birth. Alician blue and alizarin red staining revealed that the lengths of skeleton, femur and vertebrae were reduced by 21, 26 and 14%, respectively, in the knockout (KO) compared with wild type (WT) mice. In order to determine if haploid insufficiency of Osx in chondrocytes influenced postnatal skeletal growth, we compared skeletal phenotype of floxed heterozygous mice that were Cre-positive or Cre-negative. Body length was reduced by 8%, and areal BMD of total body, femur, and tibia was reduced by 5, 7, and 8% respectively in mice with conditional disruption of one allele of Osx in chondrocytes. Micro-CT showed reduced cortical vBMD and trabecular BV/TV in the femurs of Osxflox/+;col2α1-Cre mice. Histological analysis revealed that the impairment of longitudinal growth was associated with disrupted growth plates in the Osxflox/+;col2α1-Cre mice. Primary chondrocytes isolated from KO embryos showed reduced expression of chondral ossification markers, but elevated expression of chondrogenesis markers. Osx expressed in chondrocytes regulates bone growth in part by regulating chondrocyte hypertrophy."

Loss of Osterix downregulated Col10{up} and MMP13.  Loss of Osterix upregulated Fgfr3, Sox9{up}, TGFBr2, and HES1{up in LSJL}.


Osterix regulates corticalization for longitudinal bone growth via integrin β3 expression.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052162/

"Corticalization, coalescence of trabecular bone into the metaphyseal cortex, is important for the longitudinal growth of long bones. However, little is known about the molecular mechanisms controlling corticalization. To understand the molecular mechanisms underlying corticalization, we analyzed osteoblast-specific Osterix-knockout mice (Col-OMT). In control mice, corticalization was initiated after 7 postnatal days, and the number of osteoblasts in the peripheral spongiosa was increased compared to the number in the central spongiosa. In contrast, in Col-OMT mice, corticalization was delayed, and the number of osteoblasts in peripheral zones was unchanged compared to the central zone. Furthermore, femoral length was decreased in Col-OMT mice at 1 month. Because Col-OMT mice exhibited impaired matrix coalescence and osteoblast migration, we evaluated integrin signaling in Col-OMT mice. Osterix bound to the Itgb3 promoter and increased transcription of the Itgb3 gene in osteoblast cells. Interestingly, the inner and outer cortical bones were separated in Itgb3-null mice at postnatal day 7. In Itgb3-null mice, the number of osteoblasts in peripheral zones was not changed, and the femoral length was decreased. Taken together, these results indicate that Osterix regulates corticalization for longitudinal bone growth via the control of integrin β3 expression in osteoblasts. Our findings imply that the ability to control osteoblast function during corticalization may help in the treatment of short stature."

"growth plate development, trabecular bone is formed around calcified cartilage in the ossification zone in a process called endochondral bone formation. The newly formed trabecular bone coalesces at the metaphyseal cortex. This event is called “corticalization” and is the process by which longitudinal bone growth is completed⁶. The corticalization process during early growth is essential for the prediction of trabecular and cortical morphology in adulthood"

"During endochondral bone development, the bone collar, which is formed by intramembranous bone, and trabecular bone, which is formed by endochondral bone, are divided into different compartments."

The bone collar is a cuff of periosteal bone that forms around the diaphysis of the hyaline cartilage model in developing long bones

Osteoadherin

The glycosylation profile of osteoadherin alters during endochondral bone formation.

"Osteoadherin (OSAD), a keratin sulphate (KS)-substituted small leucine-rich proteoglycan has been isolated from mineralized tissues and is considered to be a mineralized tissue-specific protein. This protein [is expressed] throughout mouse metatarsal long bone development, from the cartilage anlagen (E15) to the fully formed bone (Adult). OSAD was produced with the onset of mineralization and the formation of the ossification center.  Initial OSAD localization was restricted to the endosteal surfaces of the diaphysis and forming metaphysis. OSAD was substituted with varying patterns of glycosylation during bone development. OSAD lacked any KS chains throughout all development stages. Whereas, in the mineral bound fractions, with long bone maturation the substitution with KS became more apparent with development.  Different pools of OSAD are produced during endochondral bone formation and these may have specific roles in directing the mineralization process."

"During endochondral bone development DCN was not detected in bone whereas BGN{up} was located to developing growth plates"

"DCN has a principle role in collagen type I fibril formation, binds to calcium and regulates hydroxyapatite (HAP) crystal growth"

"DCN[KO] does not exhibit a skeletal phenotype"

"During endochondral ossification FMD was localized to the developing ossification center and LUM{up} detected in pre-osteogenic tissue"

"In bone, OSAD was localized to the primary spongiosa of bovine fetal rib growth plate, and OSAD [has a] similar distribution to BSP{up} in rat long bone and calvaria"

"OSAD mRNA has been detected within mature osteoblasts and a role has been proposed in the regulation of cell proliferation and migration"

"[OSAD has] a high affinity for HAP[hydroxyapatite] via the extended acidic C-terminal and in cell attachment via the integrin αvβ3"

NMR1 strain of mice: "embryonic (E) days 15, 18/19, new born (NB), 5 days (5d) and adult (Ad (> 3 months))."

OSAD levels were low in young tissues but very high in adult tissues.  BGN increased to day 5 but levels were reduced in adult.  There was no obvious trend with Decorin and FMD was extremely elevated in adult tissues.

Aldehyde Dehydrogenase 2

Disruption of aldehyde dehydrogenase 2 gene results in altered cortical bone structure and increased cortical bone mineral density in the femoral diaphysis of mice.

"Aldehyde dehydrogenase 2 (ALDH2) degrades acetaldehyde produced by the metabolism of alcohol. The inactive ALDH2 phenotype is prevalent in East Asians, and an association between this ALDH2 polymorphism and osteoporosis has been reported. Alcohol consumption results in decreased trabecular bone volume in aldh2 knockout (aldh2(-/-)) mice compared with the volume in wild-type (aldh2(+/+)) mice.
We examined aldh2(-/-) and aldh2(+/+) mice at the ages of 4, 8 and 12week Osteogenic activities in aldh2(-/-) and aldh2(+/+) mice were assessed by osteoblast culture from calvariae of the neonatal mice. Bilateral femoral and tibial bones containing no bone marrow cells of 8-week-old mice were used for analysis of mRNA expression. mRNA expression in aldh2(-/-) and aldh2(+/+) mice after tail suspension or climbing exercise for 7days from 8weeks was analyzed to clarify the response to mechanical loading.
At 12weeks, there were no significant differences in femoral bone length, trabecular BMD in the distal metaphyses of the femurs, or mechanical strength between aldh2(-/-) and aldh2(+/)(+) mice, whereas cortical BMD and cortical thickness were significantly increased and cross-sectional area and bone marrow area were significantly decreased in the femoral diaphysis of aldh2(-/-) mice relative to the corresponding values in aldh2(+/+) mice. At 8weeks, bone formation rate and mineral apposition rate on the periosteal and endocortical surfaces were significantly increased in aldh2(-/-) mice relative to the rates in aldh(+/+) mice. Calvarial osteoblast culture study revealed that the percentage of alkaline phosphatase stained cells was significantly higher in aldh2(-/-) mice compared to that in aldh(+/+) mice. Quantitative real-time RT-PCR revealed a significant increase in the expressions of bmp2, osterix, runx2, and col1a1 mRNA in aldh2(-/-) mice, along with an increase in the expression of wnt5a mRNA and the lrp5/sost mRNA ratio. The mRNA expressions of bmp2, osterix, runx2 and pthr in aldh2(-/-) mice were significantly decreased after climbing exercise compared to those in the control, although the mRNA expressions of bmp2, osterix, runx2 were not significantly decreased and pth mRNA expression was increased in aldh(+/+) mice after climbing exercise."

"aldh2−/− mice that consume alcohol show decreased trabecular bone volume because of alcohol-induced reduction of bone formation associated with elevated p21 expression in bone marrow cells"

There was a very minor non-statistically sigificant decrease in femoral bone length in Aldh2-/- mice at 8 and 12 weeks.

What's the typical gene expression for the epiphysis of human long bones?

Since the goal of LSJL is to induce chondroinduction in the epiphysis of the long bone for new height gain.  Note that growth plates do not have to be a linear straight line like in the epiphyseal plate they can be round within the epiphysis.


Expression of genes for bone morphogenetic proteins BMP-2, BMP-4 and BMP-6 in various parts of the human skeleton.

"Seven bone samples recovered from various parts of skeletons from six cadavers of young healthy men who died in traffic accidents were collected. 

Expression of m-RNA for BMP-2{up in LSJL} and BMP-4 is higher in trabecular bone in epiphyses of long bones, cranial flat bones and corpus mandibulae then in the compact bone of diaphyses of long bones. BMP-4 was higher [in all samples] than for BMP-2.

BMP-6 is not expressed in the collected samples at all."

So if BMP-6 is important for chondrogenesis that could be a problem.  Another issue could be if BMP-2 needs to be at higher levels than BMP-4 to be permissive for cartilage growth.

"In bone matrix BMPs are connected with collagen and are not active unless released by the action of collagenases of osteoclasts"<-Thus degradation of bone matrix could play a key role in chondroinduction by LSJL.

"Biopsies (5–7 cm long and 2–3 cm wide) were taken from seven different bones. Bone marrow was removed from trabecular bone"

Prdm16

Gene expression changes in the secondary palate and mandible of Prdm16 ( -/- ) mice.

"Loss of Prdm16 expression in the mouse leads to a complete cleft of the secondary palate. We have now determined changes in gene expression in the secondary palates of Prdm16 ( -/- ) fetuses in an attempt to reveal the mechanism(s) leading to the failure of palate closure in these mice. Defined pathway-based polymerase chain reaction arrays were used to analyze the expression of genes associated with the extracellular matrix and the transforming growth factor-β and bone morphogenetic protein signaling networks, perturbations of which can lead to palatal clefting. Loss of Prdm16 expression in the secondary palate leads to alterations in numerous genes within these groups, many of which have been linked to chondrogenesis and osteogenesis. The expression of several genes linked to bone development was significantly changed in the developing secondary palate. Analysis of gene expression in the mandibles of Prdm16 ( -/- ) fetuses revealed similar alterations in the same gene set. Prdm16 [regulates] genes that play a role in the differentiation of mesenchymal cells into chondro-/osteocytes."

"PRDM16 forms a complex with both transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP)-specific Smad proteins"

"Prdm16 −/− fetuses have a smaller mandible"

Genes upregulated by PRDM16 knockout(combined data from soft palate and mandible):

Col2a1

Hapln1

MMP11

Sgce

Bmp1

GDF7

GDF6

Gsc

IL6

SHOX2(upregulated in the soft palate and downregulated in the mandile)

Col1a1

Acan

Down:

Osterix

Osteopontin

Alkaline Phasphatase

VCAM-1

VCAM1 is downregulated by LSJL.


Adiponectin and leptin induce vcam-1 expression in human and murine chondrocytes.

"VCAM-1 expression was assessed upon treatment with leptin, adiponectin and other pertinent reagents in cultured human primary chondrocytes. Signal transduction pathways have been explored by using specific pharmacological inhibitors in the adipokine-stimulated human primary chondrocytes and ATDC5 murine chondrocyte cell line.
Leptin and adiponectin increase VCAM-1 expression in human and murine chondrocytes. In addition, both adipokines have additive effect with IL-1β. Several kinases, including JAK2, PI3K and AMPK are at a play in the intracellular signalling of VCAM-1 induction."

"VCAM-1 is an inducible surface glycoprotein that belongs to immunoglobulin gene superfamily (IgSF). VCAM-1 serves as surface ligand for VLA-4 (α4β1) integrin and this adhesion molecule plays a main role in the adhesion of lymphocytes to endothelium in the site of inflammation"

"pro-inflammatory cytokines such as IL-1β and TNF-α are able to up-regulate VCAM-1 expression in primary cultures of human articular chondrocytes. VCAM-1 might contribute to adhesion of T-lymphocytes to chondrocytes, and thus participate in host defense mechanisms during inflammatory joint conditions"

"adiponectin induces NOS2, MMP-3, MMP-9 and IL-6 in chondrocytes"

"leptin and IL-1 induce synergistically nitric oxide in chondrocytes"

Patched1

Inactivation of Patched 1 in chondrocytes causes spinal fusion without inflammation.

"During development of the vertebrate skeleton chondrocytes form a cartilage template that is gradually replaced by bone. Hormones of the Hedgehog (Hh) family have been implicated in the ossification process.  During the first weeks after birth all mb1-Cre+/- /Ptch1flox/flox mice developed a progressive spinal fusion with malformation of the vertebrae. The aclampsia phenotype was caused by aberrant chondrocyte proliferation in the intervertebral discs that blocked endochondral ossification. Importantly, the disease pattern occurred in an inflammation-independent manner. Chronic activation of the Hh signal pathway in spinal chondrocytes can trigger an ankylosing spine morphology without immune cell contributions. Hence, the destruction of cartilage and loss of axial joint integrity can result from chondrocyte-intrinsic defects of monogenic origin. "

"Ligation of Ptch1 releases its inhibition of the transmembrane signaling partner Smoothened (Smo), which in turn activates intracellular signaling cascades, most notably through activation of transcription factors of the Gli family"

"Compared with wild-type mice, the tail vertebrae of Ptch1 mutants were much shorter, irregularly shaped and severely lacerated"

"These experiments revealed massive proliferation of chondrocytes and an abnormal growth plate with an absence of hypertrophic chondrocytes in the hyperproliferative cell fraction[in Ptch1 mutants]. Furthermore, staining with Safranin/Weigert to discriminate between bone and cartilage revealed the presence of proliferating chondrocytes in the intervertebral discs and a marked loss of fibrous cartilage,"<-So excess chondrocyte proliferation doesn't increase height if it comes at the expense of chondrocyte proliferation.  Maybe height would be greater if chondrocyte hypertrophy was induced after excess chondrocyte proliferation.

"The nucleus pulposus seemed to be displaced by the proliferating chondrocytes. Importantly, no infiltration of leukocytes could be detected.  These data demonstrated ongoing chondrocyte proliferation in the intervertebral discs of adult Ptch1 mutants, which in turn blocked chondrocyte maturation and proper endochondral ossification."

"Chronic activation of the chondrocytic Hh pathway following Ptch1 ablation suffices to induce spinal fusion without aberrant activation of the immune system."

"In the growth plate of the limbs, forced expression of either Shh or Ihh activates chondrocyte proliferation and impairs joint formation. In the lumbar spine, chondrocyte proliferation and hedgehog responsiveness measured by a Gli1 reporter ceases during aging. Importantly, proliferation is abolished after 3 weeks postnatally"

Dicer

LSJL downregulates Dicer1.


Dicer-dependent pathways regulate chondrocyte proliferation and differentiation.

"Dicer, an essential component for biogenesis of miRNAs, is essential for normal skeletal development. Dicer-null growth plates show a progressive reduction in the proliferating pool of chondrocytes, leading to severe skeletal growth defects and premature death of mice. The reduction of proliferating chondrocytes in Dicer-null growth plates is caused by two distinct mechanisms: decreased chondrocyte proliferation and accelerated differentiation into postmitotic hypertrophic chondrocytes. These defects appear to be caused by mechanisms downstream or independent of the Ihh-PTHrP signaling pathway, a pivotal signaling system that regulates chondrocyte proliferation and differentiation. Microarray analysis of Dicer-null chondrocytes showed limited expression changes in miRNA-target genes, suggesting that, in the majority of cases, chondrocytic miRNAs do not directly regulate target RNA abundance.Dicer-dependent pathway [regulates] chondrocyte proliferation and differentiation during skeletal development"

"primiRNAs are cleaved into small-hairpin premiRNAs by the microprocessor complex containing Drosha and DGCR8. premiRNAs are exported into the cytoplasm, where the RNase III, Dicer, removes the loop region of the hairpin."

Lack of dicer downregulates miR-30c and let-7b.

"At E18.5, the hypertrophic region, indicated by Col10a1 expression, was expanded, whereas the Mmp13 expression domain of the cartilage was not expanded in Col2-Cre:Dicerfl/fl mice. Because Mmp13 is expressed only by terminally differentiated hypertrophic chondrocytes in the growth plate, these observations suggest that the expansion of the hypertrophic region was caused by an acceleration of hypertrophic differentiation of proliferating chondrocytes rather than a reduction in cartilage resorption by bone cells, which would cause an increase in terminally differentiated hypertrophic chondrocytes. Expansion of the hypertrophic region can also occur as a consequence of stimulation of chondrocyte differentiation at earlier steps"

"We did find up-regulation of Hmga2 mRNA in Dicer-deficient chondrocytes."<-HMGA2 is upregulated by LSJL.  Tamoxifen inhibits dicer.  Dicer inhibition also upregulated COL10A1 and PTH-R.

Gene comparison of LSJL to gene info given in Table3 to be done.

Table4 provides a very interesting list of miRNA binding sites in chondrocytes.

Chitanase 3-like protein's

LSJL downregulates CHI3L3.

Human YKL39 (chitinase 3-like protein 2), an osteoarthritis-associated gene, enhances proliferation and type II collagen expression in ATDC5 cells.

"Human YKL39 (chitinase 3-like protein 2 /CHI3L2) is a secreted 39 kDa protein produced by articular chondrocytes and synoviocytes. hYKL39 expression is increased in osteoarthritic articular chondrocytes. Sequence analyses indicated that hYKL39 has significant identity with the human chitotorisidase family molecules, although it is considered that hYKL39 has no enzymatic activity since it lacks putative chitinase catalytic motif. We overexpressed hYKL39 in ATDC5{chondrocyte progenitor cell line} cells. hYKL39 enhances colony forming activity, cell proliferation, and type II collagen expression in these cells."

"overexpression of hYKL39 enhanced cell proliferation by around 3-fold [and increased the percentage of cells in the S-phase]"

"expression of Cyclin D1, whose expression level is known to increase transiently in late G1 phase, was significantly increased in hYKL39 overexpressing cells"

"DDR1 expression is significantly increased in the hYKL39 overexpressing cells while integrin beta-1 and beta-3 expression was significantly decreased"

"Chitin is a long-chain biopolymer of N-acetylglucosamine (GlcNAc) similar to the structure of cellulose"

Lactate

Lactate is lactic acid.

Lactate Influences the Gene Expression Profile of Human Mesenchymal Stem Cells (hMSC) in a Dose Dependant Manner.

"Lactate [stimulates] collagen synthesis and vessel growth. Lactate, in vivo, plays an important role in homing of stem cells. hMSCs were obtained from bone marrow. Subsequently the hMSCs were treated with either 0, 5, 10 and 15 mM lactate (pH 7,4) for 24 hours. RNA Isolation from stimulated hMSCs and controls was performed. Lactate in moderate concentrations of 5 respectively 10 mM leads to an anti-inflammatory, anti-apoptotic but growth and proliferation promoting gene expression after 24 h. In contrast, high lactate concentrations of 15 mM leads to the opposed effect, namely promoting inflammation and apoptosis. Hypoxia induced genes did not show any significant regulation. Contrary to expectation, we were not able to show any significant regulation of candidates associated with glycolysis."

TGF-Beta was only upregulated 1.794 fold 10 mM and not 5 and 15 fold.  VEGF-A was upregulated at 10 and 15 mM but VEGF-B was only upregulated at 15 mM.  Expression of the pro-chondrogenic HIF-1a peaked at 10 mM but was not statistically significant.


Lactate modulates gene expression in human mesenchymal stem cells.

"Surgical wounds are characterised by elevated tissue lactate concentrations. This accumulated lactate is capable of stimulating collagen synthesis and new vessel growth as well. Lactate is  able to favour homing of stem cells.

MSC were isolated from human bone marrow using the density gradient technique and incubated with alpha-methoxyethoxymethyl containing 10% fetal calf serum at 37 degrees C under 95% air and 5% CO(2). MSC were treated with 15 mM lactate for different time periods (1, 6 and 24 h and 3 and 7 days). A significant alteration of gene expression was defined as a two-fold stimulation or inhibition. 

Gene expression analysis shows 63 up- and 51 down-regulated genes after 1 h of treatment, 45 up- and 47 down-regulated genes after 6 h of treatment, 57 up- and 72 down-regulated genes after 24 h of treatment, 103 up- and 28 down-regulated genes after 3 days of treatment and 50 up- and 101 down-regulated genes after 7 days of treatment with lactate. The majority of the modulated genes are related to the expression of cytokines, transcription factors and cell-cycle- or cellular-matrix-associated proteins. In particular, lactate up-regulates the expression of interleukin-6{up} (3 days, 4.11-fold), of heat shock protein 70 (3 days, 2.36-fold) and of hypoxia-inducible factor-1alpha (3 days, 2.09-fold). A down-regulating effect of lactate is observed for superoxide dismutase 2 (1 h, 0.5-fold; 24 h, 0.4-fold; 7 days, 0.32-fold) and BCL2-associated X protein (24 h, 0.42-fold; 7 days, 0.4-fold). Expression of cell surface antigens clusters of differentiation 29, 44, 59, 73, 90, 105, 106 and 146 does not change over the time period of lactate treatment."