Increase Height with Indian Hedehog proteins

The Indian Hedgehog protein plays a vital role in endochondral ossification(long bone growth).  It helps to regulate many growth plate processes. 

Recapitulation of endochondral bone formation using human adult mesenchymal stem cells as a paradigm for developmental engineering. 

[Recapitulation refers to replicating.  So they're trying to replicate long bone growth using mesenchymal stem cells.] 

"Mesenchymal stem/stromal cells (MSC) are typically used to generate bone tissue by a process resembling intramembranous ossification, i.e., by direct osteoblastic differentiation. However, most bones develop by endochondral ossification, i.e., via remodeling of hypertrophic cartilaginous templates[the growth plate]. To date, endochondral bone formation has not been reproduced using human, clinically compliant cell sources. We aimed at engineering tissues from bone marrow-derived, adult human MSC with an intrinsic capacity to undergo endochondral ossification[By definition the cells in your bone marrow have the capacity to undergo endochondral ossification so if you get new MSCs to differentiate into chondrocytes by a method such as LSJL...]. By analogy to embryonic limb development, we hypothesized that successful execution of the endochondral program depends on the initial formation of hypertrophic cartilaginous templates[And what causes this initial formation?  Mesenchymal stem cells]. Human MSC, subcutaneously implanted into nude mice at various stages of chondrogenic differentiation, formed bone trabeculae only when they had developed in vitro hypertrophic tissue structures. Advanced maturation in vitro resulted in accelerated formation of larger bony tissues. The underlying morphogenetic process was structurally and molecularly similar to the temporal and spatial progression of limb bone development in embryos. In particular, Indian hedgehog signaling was activated at early stages and required for the in vitro formation of hypertrophic cartilage. Subsequent development of a bony collar in vivo was followed by vascularization, osteoclastic resorption of the cartilage template, and appearance of hematopoietic foci[Vascularization may inhibit height growth by inhibiting hypoxia; note this is not fusion but merely osteoclasts cleaning up left over ECM]. This study reveals the capacity of human MSC to generate bone tissue via an endochondral program and provides a valid model to study mechanisms governing bone development. Most importantly, this process could generate advanced grafts for bone regeneration by invoking a "developmental engineering" paradigm." 

So Mesenchymal Stem cells can be used for bone regeneration(i.e. to cause a new growth plate) and mesenchymal stem cells may be closely linked to the Indian Hedgehog protein. 

"adult expanded human MSC [can] generate de novo bone tissue in vivo through endochondral ossification{adult MSC's are able to undergo endochondral ossification and help you grow taller}. The observed endochondral morphogenesis bears striking features of normal endochondral ossification as is typical for limb skeletal development, namely, (i) cellular condensation and hypertrophic chondrogenesis, (ii) functional dependence on IHH signaling, (iii) formation of a bony collar by perichondral ossification, (iv) MMP-mediated matrix remodeling, vascularization, and osteoclastic activity, (v) bone matrix deposition over the resorbed cartilaginous template, and (vi) formation of complete bone tissue, which likely includes functional hematopoietic foci."

Atf4 regulates chondrocyte proliferation and differentiation during endochondral ossification by activating Ihh transcription. 

"Activating transcription factor 4 (Atf4) is a leucine-zipper-containing protein of the cAMP response element-binding protein (CREB) family. Ablation of Atf4 (Atf4(-/-)) in mice leads to severe skeletal defects, including delayed ossification and low bone mass, short stature and short limbs. Atf4 is expressed in proliferative and prehypertrophic growth plate chondrocytes, suggesting an autonomous function of Atf4 in chondrocytes during endochondral ossification. In Atf4(-/-) growth plate, the typical columnar structure of proliferative chondrocytes is disturbed. The proliferative zone is shortened, whereas the hypertrophic zone is transiently expanded. The expression of Indian hedgehog (Ihh) is markedly decreased, whereas the expression of other chondrocyte marker genes, such as type II collagen (Col2a1), PTH/PTHrP receptor (Pth1r) and type X collagen (Col10a1), is normal. Furthermore, forced expression of Atf4 in chondrocytes induces endogenous Ihh mRNA{Injecting atf4 forces the body to produce indian hedgehog}, and Atf4 directly binds to the Ihh promoter and activates its transcription. reactivation of Hh signaling pharmacologically in mouse limb explants corrects the Atf4(-/-) chondrocyte proliferation and short limb phenotypes. ATF4 [is a] transcriptional activator of Ihh in chondrocytes that paces longitudinal bone growth by controlling growth plate chondrocyte proliferation and differentiation." 

Indian Hedgehog is necessary for maximizing height "potential" but it's unclear whether it's possible for their to be a mutation on Indian Hedgehog to induce supernatural growth.   Atf4 controls Ihh which in turn controls growth plate chondrocyte proliferation and differentiation.

"Atf4 is expressed in proliferative and prehypertrophic growth plate chondrocytes, suggesting an autonomous function of Atf4 in chondrocytes during endochondral ossification. In Atf4(-/-) growth plate, the typical columnar structure of proliferative chondrocytes is disturbed. The proliferative zone is shortened, whereas the hypertrophic zone is transiently expanded. The expression of Indian hedgehog (Ihh) is markedly decreased, whereas the expression of other chondrocyte marker genes, such as type II collagen (Col2a1), PTH/PTHrP receptor (Pth1r) and type X collagen (Col10a1), is normal. forced expression of Atf4 in chondrocytes induces endogenous Ihh mRNA, and Atf4 directly binds to the Ihh promoter and activates its transcription."

"Ihh can also diffuse and reach cells in the articular perichondrium, where it induces the expression of parathyroid hormone related protein (PTHrP; Pthlh — Mouse Genome Informatics), which in turn inhibits chondrocyte hypertrophy and maintains the pool of proliferative chondrocytes"

Indian Hedgehog is partially what keeps chondrocytes from hypertrophying in the articular cartilage(and therefore ossifying).  In turn Atf4 plays a role as well as it is involved in Ihh transcription.

"Runx2, with the assistance of Runx3, binds directly to the Ihh promoter and activates its expression"

Zfp521 increases height and inhibits Runx2 activity. But Atf4 deletion decreases height.  Therefore, Ihh, Atf4, and Runx2 go in the category of compounds that you need a balance of.

Morphology and physiology of the epiphyseal growth plate. 

"The epiphyseal growth plate develops from the cartilaginous-orientated mesenchymal cells that express SOX family genes[mesenchymal stem cells with the SOX family genes have the ability to create a new growth plate]. This multilayer structure is formed by the proliferation and hypertrophy of cells that synthesize the extracellular matrix composed of collagen (mainly type II, IX, X, XI) and proteoglycans (aggrecan, decorin, annexin II, V and VI). The resting zone is responsible for protein synthesis and maintaining a germinal structure. In the proliferative zone, cells rapidly duplicate. The subsequent morphological changes take place in the transformation zone, divided into the upper and lower hypertrophic layers. In the degenerative zone, the mineralization process becomes intensive due to increased release of alkaline phosphate, calcium and matrix vesicles by terminally differentiated chondrocytes and some other factors e.g., metaphyseal ingrowth vessels. At this level, as well as in the primary and secondary spongiosa zones, chondrocytes undergo apoptosis and are physiologically eliminated. Unlike adult cartilage, in fetal and early formed growth plates, unusual forms such as authophagal bodies, paralysis and dark chondrocytes were also observed. Their ultrastructure differs greatly from apoptotic and normal cartilage cells. Chondrocyte proliferation and differentiation are regulated by various endocrine, paracrine, and autocrine agents such as growth, thyroid and sex hormones, beta-catenin, bone morphogenetic proteins, insulin-like growth factor, iodothyronine deiodinase, leptin, nitric oxide, transforming growth factor beta and vitamin D metabolites[Even if Growth is dependent on Mesenchymal Stem Cells it is still possible to maximize your growth by optimizing chondrocyte differentiation and proliferation per stem cell]. However, the most significant factor is parathyroid hormone-related protein (PTHrP) which is synthesized in the perichondrium by terminally differentiated chondrocytes. Secondary to activation of PTH/PTHrP receptors, PTHrP stimulates cell proliferation by G protein activation and delays their transformation into prehypertrophic and hypertrophic chondrocytes. When proliferation is completed, chondrocytes release Indian hedgehog (Ihh), which stimulates PTHrP synthesis via a feedback loop. Any disturbances of the epiphyseal development and its physiology result in various skeletal abnormalities known as dysplasia." 

So to maximize growth. First we want to increase the number of mesenchymal stem cells by maximizing mesenchymal stem cell proliferation. Then we want to maximize chondrocyte proliferation and differentation that is induced per stem cell with methods like those involving hormone manipulation. Then we want to make sure that everything is properly expressed like Indian Hedgehog.

A possible Indian Hedgehog mutation that might induce height growth would be one that causes hyper PTHrP synthesis. Remember one theory of growth plate activity activity is that it's a war between chondrogenesis and osteogenesis[perhaps more acurately a ware between chondrogenesis and vascularization]. You want to make sure chondrogenesis is winning at all times. This osteogenesis occurs in the perichondrium by apoptosis of chondrocytes. These dead chondrodcytes release IHH which causes the parathyroid to release a protein that stimulates chondrocyte differentiation. A mutation that causes hyper PTHrP expression may cause you to grow forever barring a sufficient supply of stem cells to differentiate into chondrocytes.

"The initial steps in cartilage formation are secondary to condensation of mesenchymal cell from 12 to 15 per 1000 μm2{you need at least 12 to 15 mesenchymal stem cells to condensate to grow taller with LSJL}. MPCs initiate chondrogenesis by first migration to presumptive skeletogenic sites from the cranial neural crest, paraxial mesoderm, and lateral plate mesoderm and formation of cell mass condensations. MPCs divide in the center of the condensations to form prechondrocytes that turn off the expression of mesenchymal and condensation markers. Instead of an elongated shape they become rounder with concomitant decrease of intercellular adhesion and intensive endothelial cell proliferation"

"Ihh synthesis begins throughout mesenchymal condensation, increases during cartilage formation and later decreases. Without the factor[Ihh], the perichondrium transforms into periosteum"

Ihh may inhibit the aging of the perichondrium into the periosteum.

Indian hedgehog gene transfer is a chondrogenic inducer of human mesenchymal stem cells.

"First generation adenoviral vectors encoding the cDNA of the human IHH gene were created by cre-lox recombination and used alone or in combination with adenoviral vectors, bone morphogenetic protein- 2 (Ad. BMP-2), or transforming growth factor beta-1 (Ad. TGF-beta1) to transduce human bone-marrow derived MSCs at 5 x 102 infectious particles/cell. Thereafter, 3 x 105 cells were seeded into aggregates and cultured for 3 weeks in serum-free medium, with untransduced or marker gene transduced cultures as controls.
IHH, TGF-beta1 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs as evidenced by strong staining for proteoglycans, collagen type II, increased levels of glycosaminoglycan (GAG) synthesis, and expression of mRNAs associated with chondrogenesis{If IHH can induce chondrogenesis as effectively as TGF-Beta1 and BMP-2 then we should probably trying to induce increased IHH expression as strongly as we are the latter two proteins}. IHH-modified aggregates, alone or in combination, showed a tendency to progress towards hypertrophy, as judged by the expression of alkaline phosphatase and stainings for collagen type X and Annexin 5."

The study mentions that Sox9 is a downstream regulator of Shh.

"major differences between the different chondrogenic groups IHH, IHH + TGF-β1, or IHH + BMP-2
could not be detected."<-So maybe IHH is sufficient to induce chondrogenesis on it's own.

"the transcription factor Alf4 was shown to be required in chondrocytes for IHH expression"

"IHH was shown be able to effectively induce and regulate several steps during chondrogenesis, including proliferation, differentiation, hypertrophy and apoptosis"

BMP-2+Ihh group had more hypertrophic chondrocyte factors than other groups.

"tissue culture using complete DME Medium (DMEM) containing 10% FBS and 1% penicillin/streptomycin, 1 ng/mL FGF-2"<-So FGF-2 was required in addition to IHH.

Clock genes influence gene expression in growth plate and endochondral ossification in mice.

"[There is] transient promotion by parathyroid hormone of Period-1 (Per1) expression in cultured chondrocytes. The clock genes of chondrogenic differentiation [are modulated] through gene transactivation of Indian hedgehog (Ihh) in chondrocytes of the growth plate. Several clock genes were expressed with oscillatory rhythmicity in cultured chondrocytes and rib growth plate in mice, while chondrogenesis was markedly inhibited in stable transfectants of Per1 in chondrocytic ATDC5 cells and in rib growth plate chondrocytes from mice deficient of brain and muscle aryl hydrocarbon receptor nuclear translocator-like (Bmal1). Ihh promoter activity was regulated by different clock gene products, with clear circadian rhythmicity in expression profiles of Ihh in the growth plate. In Bmal1-null mice, a predominant decrease was seen in Ihh expression in the growth plate with a smaller body size than in wild-type mice. Bmal1 deficit led to disruption of the rhythmic expression profiles of both Per1 and Ihh in the growth plate. A clear rhythmicity was seen with Ihh expression in ATDC5 cells exposed to dexamethasone. In young mice defective of Bmal1 exclusively in chondrocytes, similar abnormalities were found in bone growth and Ihh expression."

"constitutive mRNA expression was seen for all clock genes examined irrespective of culture durations of 7 or 21 days on analysis performed at 32 cycles [including] Bmal1, Clock, Per1, Per2, Per3, Cry1, Cry2, the Basic helix-loop-helix proteins differentiated embryo chondrocyte 1 (Dec1){up in LSJL as bhlhb2} and Dec2, D site of albumin promoter binding protein (Dbp), and Rev-erbα"

"Period proteins are known to be synthesized through gene transactivation mediated by the positive Bmal1/Clock complex at upstream E-box elements, but to inhibit the gene transactivation as a negative feedback loop together with Cry proteins after the accumulation in the cytoplasm for subsequent translocation into the nucleus. Both Per and Cry repressor members are poly-ubiquitinylated for degradation by the proteasome pathway, which in turn releases the inhibition by the negative Per/Cry complex of gene transactivation mediated by the positive Bmal1/Clock complex. This positive and negative feedback loop occurs at 24 h-cycle rhythmicity" Maybe due to the cyclical nature of these genes they just weren't present at the time the gene expression analysis is done but that does not mean they are not involved in LSJL.

"Per1 would interfere with rhythmic promotion by the Bmal1/Clock complex of the transcription of Ihh at the upstream E-box element in growth plate chondrocytes toward disturbance of chondrogenesis. Accordingly, rhythmic expression of Ihh could lead to oscillated rhythmic bone elongation through endochondral ossification during skeletogenesis"

"the present results obtained in Bmal1-/- mice argue in favor of an idea that longitudinal bone elongation would be concurrently regulated by the rhythmic expression of clock gene products by chondrocytes"

Activation of the Hh pathway in periosteum-derived mesenchymal stem cells induces bone formation in vivo: implication for postnatal bone repair.

"Using a murine segmental bone graft transplantation model, we isolated a population of early periosteum-callus-derived mesenchymal stem cells (PCDSCs) from the healing autograft periosteum. These cells express typical mesenchymal stem cell markers and are capable of differentiating into chondrocytes. Activation of the hedgehog (Hh) pathway effectively promoted osteogenic and chondrogenic differentiation of PCDSCs in vitro and induced bone formation in vivo. To determine the role of the Hh pathway in adult bone repair, we deleted Smoothened (Smo) {upregulated by LSJL}, the receptor that transduces all Hh signals at the onset of bone autograft repair via a tamoxifen-inducible RosaCreER mouse model. We found that deletion of Smo markedly reduced osteogenic differentiation of isolated PCDSCs and resulted in a near 50% reduction in periosteal bone callus formation at the cortical bone junction."

"The insertion of the LacZ gene into the Ptc1 locus disrupts Ptc1 expression. "

"Ptc1-LacZ was detected at the early stage of healing in periosteal cells overlying the bone surface on day 3. At days 5 and 7, Ptc-LacZ–positive cells were found in periosteal callus surrounding prehypertrophic chondrocytes. The staining was markedly increased in chondroprogenitors, proliferating chondrocytes".<-so the lack of significant ptc1 expression is consistent with chondrogenesis.

"The long bones of Ihh mutants are only 20% the length of those of their wild-type littermates at birth, a phenotype that correlates with a pronounced reduction in cellular proliferation."

Ihh enhances differentiation of CFK-2 chondrocytic cells and antagonizes PTHrP-mediated activation of PKA.

"Indian Hedgehog (Ihh), a member of the hedgehog (HH) family of secreted morphogens, and parathyroid hormone-related peptide (PTHrP) are key regulators of cartilage cell (chondrocyte) differentiation. We have investigated, in vitro, the actions of HH signalling and its possible interplay with PTHrP using rat CFK-2 chondrocytic cells. Markers of chondrocyte differentiation [alkaline phosphatase (ALP) activity, and type II (Col2a1) and type X collagen (Col10a1) expression] were enhanced by overexpression of Ihh or its N-terminal domain (N-Ihh), effects mimicked by exogenous administration of recombinant N-terminal HH peptide. a missense mutation mapping to the N-terminal domain of Ihh (W160G) reduces the capacity of N-Ihh to induce differentiation. Prolonged exposure of CFK-2 cells to exogenous N-Shh (5x10(-9) M) in the presence of PTHrP (10(-8) M) or forskolin (10(-7) M) resulted in perturbation of HH-mediated differentiation.  overexpression of a constitutively active form of the PTHrP receptor (PTHR1 H223R) inhibited Ihh-mediated differentiation, implicating activation of protein kinase A (PKA) by PTHR1 as a probable mediator of the antagonistic effects of PTHrP. overexpression of Ihh/N-Ihh or exogenous treatment with N-Shh led to dampening of PTHrP-mediated activation of PKA. Ihh harbors the capacity to induce rather than inhibit chondrogenic differentiation, that PTHrP antagonizes HH-mediated differentiation through a PKA-dependent mechanism and that HH signalling, in turn, modulates PTHrP action through functional inhibition of signalling by PTHR1 to PKA."

"All known HH members are proteolytically processed through an autocatalytic mechanism to generate secreted peptides corresponding to the N- and C-terminal domains of the native protein. The C-terminal domain is believed to possess the catalytic properties required for HH cleavage, which occurs at a conserved Gly-Cys site. Cholesterol, participating as a nucleophile in the autocatalytic process, attaches to the C-terminal end of the nascent N-terminal domain and enhances its lipophilic properties. The N-terminal domain is believed to possess all the known biological activities of HH proteins and is highly conserved and interchangeable amongst HH family members"<-LSJL upregulates Dhh instead of Ihh but the N-terminal domain is interchangable.

"This domain can bind its cognate receptor Patched (Ptc), a 12-transmembrane (TM) protein that otherwise interacts with, and thereby inhibits, the 7-TM receptor protein Smoothened (Smo). The ligand-induced release of Smo from its interaction with Ptc results in intracellular signal transduction."

"Treatment of bone explants from wild-type mice with Sonic Hedgehog (Shh) protein mimicked the ability of PTHrP to inhibit Col10a1 expression"

"N-Ihh W160G may be capable of transmitting sufficient levels of HH-signalling to selectively induce certain markers of differentiation (Col10a1) but not others (ALP, Col2a1)"

"the W160G mutation reduces the capacity of N-terminal Ihh to stimulate Ptc expression, indicative of a partial loss in HH-signalling activity."

"Dose-response induction of ALP, Col2a1 and Col10a1 by exogenous N-Shh"

"In bone cells, PTHrP mediates most of its biological actions through activation of its cognate G-protein-coupled receptor, PTHR1, leading to stimulation of adenylate cyclase and consequent PKA activation. PKA has been widely described as an inhibitor of HH-signalling in multiple systems"

"PTHrP led to severe dampening of HH-induced Col2a1 and Col10a1 mRNA expression. In contrast, PTHrP had no effect on N-Shh-induced Ptc expression. Moreover, all the antagonistic effects exerted by PTHrP on HH-action were also mimicked by forskolin suggesting that this phenomenon may be attributed to PKA activation"

"phosphorylation of Gli3, but not Gli1, by PKA results in its proteolysis and formation of an alternate repressor form that downregulates transcription of certain HH target genes"


Ihh and runx2/runx3 signaling interact to coordinate early chondrogenesis: a mouse model.

"we utilized the maternal transfer of 5E1 to E12.5 in mouse embryos, a process that leads to an attenuation[weakening] of Ihh activity. As a result, mouse limb bud chondrogenesis was inhibited, and an exogenous recombinant IHH protein enhanced the proliferation and differentiation of mesenchymal cells. Analysis of the genetic relationships in the limb buds suggested a more extensive role for Ihh and Runx genes in early chondrogenesis. The transfer of 5E1 decreased the expression of Runx2 and Runx3, whereas an exogenous recombinant IHH protein increased Runx2 and Runx3 expression.  Transcription factor Gli1 in hedgehog pathway enhances the direct induction of both Runx2 and Runx3 transcription."

"6-week- old mice delivered from pregnant mice injected with 5E1 at E12.5 exhibited limb, skull, tail, and trunk bones that were reduced in size and length, whereas all mice delivered from pregnant mice injected with PBS as a control showed the same bone lengths and sizes as those of wild-type mice"

"Mice treated with 5E1 at E14.5, E16.5 and E18.5 did not exhibit shorter limbs"

" Ihh signaling may be associated with the condensation of mesenchymal cells and their differentiation into chondrocytes to form the cartilage primordium."

" Ptch1 and Hhip, direct targets of hedgehog signaling, were significantly down-regulated in 5E1-treated limb buds"<-Gli1 was slightly downregulated.

" expression levels of Gli2, Gli3{up} and Smo, which are not activated in response to hedgehog signaling but are involved in the transcription of hedgehog signal genes, were not changed following 5E1 injection"<-Ihh expression also increased.

Sox9 and HHip expression also decreased and are both upregulated by LSJL.

"E12.5 to E14; [is the] period when mesenchymal cell condensation, proliferation, and differentiation into chondrocytes occur to form the cartilage primordia."

"exogenous IHH protein caused mesenchymal cells to produce more cartilage nodules compared to PBS treatment"

Genes upregulated by 5E1 transfer to E12.5 also upregulated by LSJL:

Stk36

Id2{down}

Smo

Downregulated:

Sdc4{up}

Hedgehog-Gli activators direct osteo-chondrogenic function of bone morphogenetic protein toward osteogenesis in the perichondrium.

"Rat end-plate chondrocytes were cultured and ICMT (strain at 0.5 Hz sinusoidal curve at 10% elongation) applied for 7 days for 4 h/day and cultured for a further 2 days. End-plate chondrocytes were also exposed to 10 ng/mL of TGF-β1. Then, using small interfering RNA technology, small interfering TGF-β1 (siTGF-β1) was transfected. Expression of ENPP-1 and TGF-β1 was measured.
Expression of both ENPP-1 and TGF-β1 was up-regulated after ICMT. Both RT-PCR and western blot showed that ENPP-1 expression decreases with siRNA TGF-β1 after 3% elongation 40 min, and cultured for an additional 2 days.
Down-regulation of ENPP-1 gene expression induced by ICMT is likely dependent on TGF-β1 in end-plate chondrocytes."

"During endochondral ossification, cells in the perichondrium give rise to osteoblast precursors. Hedgehog (Hh) and bone morphogenetic protein (BMP) are suggested to regulate the commitment of
these cells."

"Hh signaling suppressed BMP-induced chondrogenic differentiation; Gli1 inhibited the expression of Sox5, Sox6, and Sox9 as well as transactivation by Sox9. Ectopic expression of chondrocyte maker genes were observed in the perichondrium of metatarsals in Gli1-/- fetuses, and the phenotype was more severe in Gli1-/-;Gli2-/- newborns."

"Perichondrial cells constitute the perichondrium, a thin layer of fibroblastic cells surrounding the cartilage mold."

"forced activation of BMP signaling has been shown to cause ectopic chondrocytes in the perichondrium"

"We used SAG, a Hh signaling activator; cyclopamine, a Hh signaling inhibitor; rhBMP2; dorsomorphin, a BMP signaling inhibitor; lithium chloride (LiCl), a GSK3 inhibitor; and IWP2, an inhibitor of Wnt processing and secretion"

"SAG induced ectopic bone collar formation in the perichondrium and inhibited cartilage
mineralization; cyclopamine blocked bone collar formation and accelerated cartilage mineralization. rhBMP2 did not induce ectopic bone collar formation, but enhanced chondrogenesis in the growth plate; dorsomorphin did not affect osteogenesis, but inhibited chondrogenesis. LiCl promoted bone collar formation in synchronization with cartilage mineralization; IWP2 inhibited bone collar formation."

So HH signaling inhibition and GSK3 Beta inhibition is possibly pro cartilage.  BMPs are pro cartilage. 

"SAG suppressed rhBMP2-induced mRNA expression of chondrocyte marker genes including Col2a1, Col9a1, and Chondromodulin 1 (Chm1)"

"In C3H10T1/2 cells, Gli1 overexpression suppressed the rhBMP2-induced Col2a1 mRNA expression, whereas this suppression was not observed in response to the overexpressions of Gli2, the full length form of Gli3, or the repressor form of Gli3"<-LSJL upregulates Gli3.

The Effects of Inhibiting Hedgehog Signaling Pathways by Using Specific Antagonist Cyclopamine on the Chondrogenic Differentiation of Mesenchymal Stem Cells.

"This study aimed to investigate the effects of cyclopamine, a specific inhibitor of Hedgehog signaling pathways, on the chondrogenic differentiation of mesenchymal stem cells (MSCs). During culture, the experimental groups were treated with cyclopamine and their cell proliferation status was assessed using the MTT test. The extra-bone cellular matrix (ECM) and Collagen II (Col II) was detected by toluidine blue staining and immunohistochemistry of cells. The concentrations of Col II and aggrecan in the culture solution and cytosol were detected using ELISA on the 7th, 14th, and 21st days of cyclopamine induction. Gene and protein expression of Col II and aggrecan were analyzed on the 14th day of cyclopamine induction using real-time PCR and western blot analyses. No significant differences in proliferation of mesenchymal stem cells were found between the control group and the group treated with cyclopamine. Compared to the blank control group, the ECM level was low and the protein and mRNA concentrations of Collagen II (Col II) and aggrecan in the culture solution and cytosol, respectively, were significantly reduced in the experimental group. The Smo acted as a key point in the regulations of Hedgehog signaling pathway on the chondrogenic differentiation of rabbit MSCs."

"At the early phase of fracture repair, expression of Shh and Glil has been found in the periosteum and bone marrow cavity of the fracture site"

Indian hedgehog signaling promotes chondrocyte differentiation in enchondral ossification in human cervical ossification of the posterior longitudinal ligament.

"Histological, immunohistochemical, and immunoblot analysis of the expression of Indian hedgehog (Ihh) signaling in human cervical ossification of the posterior longitudinal ligament (OPLL).  Ihh signaling in correlation with Sox9 and parathyroid related peptide hormone (PTHrP) facilitates chondrocyte differentiation in enchondral ossification process in human cervical OPLL.Summary of Background Data. In enchondral ossification, certain transcriptional factors regulate cell differentiation. OPLL is characterized by overexpression of these factors and disturbance of the normal cell differentiation process. Ihh signaling is essential for enchondral ossification, especially in chondrocyte hypertrophy.  Samples of ossified ligaments were harvested from 45 patients who underwent anterior cervical decompressive surgery for symptomatic OPLL, and 6 control samples from cervical spondylotic myelo/radiculopathy patients without OPLL. The harvested sections were examined. The ossification front in OPLL contained chondrocytes at various differentiation stages, including proliferating chondrocytes in fibrocartilaginous area, hypertrophic chondrocytes around the calcification front, and apoptotic chondrocytes near the ossified area. Immunoreactivity for Ihh and Sox9 was evident in proliferating chondrocytes, and PTHrP was strongly positive in hypertrophic chondrocytes. Mesenchymal cells with blood vessel formation were positive for Ihh, PTHrP, and Sox9. Cultured cells from OPLL tissues expressed significantly higher levels of Ihh, PTHrP, and Sox9 compared with non-OPLL cells. Overexpression of Ihh signaling promotes abnormal chondrocyte differentiation in enchondral ossification, and enhances bone formation in OPLL."

Couldn't get full study

Inter-dependent tissue growth and Turing patterning in a model for long bone development.

"we developed a regulatory model for the core signalling factors IHH, PTCH1, and PTHrP and included two cell types, proliferating/resting chondrocytes and (pre-)hypertrophic chondrocytes. We show that the reported IHH-PTCH1 interaction gives rise to a Schnakenberg-type Turing kinetics, and that inclusion of PTHrP is important to achieve robust patterning when coupling patterning and tissue dynamics. The model reproduces relevant spatiotemporal gene expression patterns, as well as a number of relevant mutant phenotypes. In summary, we propose that a ligand-receptor based Turing mechanism may control the emergence of patterns during long bone development, with PTHrP as an important mediator to confer patterning robustness when the sensitive Turing system is coupled to the dynamics of a growing and differentiating tissue. We have previously shown that ligand-receptor based Turing mechanisms can also result from BMP-receptor, SHH-receptor, and GDNF-receptor interactions, and that these reproduce the wildtype and mutant patterns during digit formation in limbs and branching morphogenesis in lung and kidneys. Receptor-ligand interactions may thus constitute a general mechanism to generate Turing patterns in nature."

" Hypertrophic chondrocytes secrete the protein IHH, and proliferating chondrocytes express the IHH receptor PTCH1 (gene Ptch1), as well as the diffusible, extracellular protein PTHrP (gene Pthrp). IHH signalling, which results from the binding of IHH to its receptor PTCH1, enhances PTHrP synthesis. PTHrP production is also stimulated by BMP signalling. PTHrP inhibits the differentiation of proliferating chondrocytes into hypertrophic chondrocytes. The proliferation rate of the proliferating chondrocytes is enhanced by IHH signalling"

"While IHH signalling indeed enhances Pthrp expression, PTHrP signalling negatively impacts on its own expression"

" signalling in periochondrial cells is regulated by tension, which in turn indirectly impacts on bone growth"