Regulation Of Chondrocyte Proliferation Research Articles

Identification of novel genes including NAV2 associated with isolated tall stature.

Very tall people attract much attention and represent a clinically and genetically heterogenous group of individuals. Identifying the genetic etiology can provide important insights into the molecular mechanisms regulating linear growth. We studied a three-generation pedigree with five isolated (non-syndromic) tall members and one individual with normal stature by whole exome sequencing; the tallest man had a height of 211cm. Six heterozygous gene variants predicted as damaging were shared among the four genetically related tall individuals and not present in a family member with normal height. To gain insight into the putative role of these candidate genes in bone growth, we assessed the transcriptome of murine growth plate by microarray and RNA Seq. Two (Ift140, Nav2) of the six genes were well-expressed in the growth plate. Nav2 (p-value 1.91E-62) as well as Ift140 (p-value of 2.98E-06) showed significant downregulation of gene expression between the proliferative and hypertrophic zone, suggesting that these genes may be involved in the regulation of chondrocyte proliferation and/or hypertrophic differentiation. IFT140, NAV2 and SCAF11 have also significantly associated with height in GWAS studies. Pathway and network analysis indicated functional connections between IFT140, NAV2 and SCAF11 and previously associated (tall) stature genes. Knockout of the all-trans retinoic acid responsive gene, neuron navigator 2 NAV2, in Xenopus supports its functional role as a growth promotor. Collectively, our data expand the spectrum of genes with a putative role in tall stature phenotypes and, among other genes, highlight NAV2 as an interesting gene to this phenotype.

Long non-coding RNA PMS2L2 is down-regulated in osteoarthritis and inhibits chondrocyte proliferation by up-regulating miR-34a

Long non-coding RNA (lncRNA) PMS2L2 has been reported to participate in endotoxin-induced inflammatory responses. As these types of responses can promote osteoarthritis (OA), it was of interest to ascertain if PMS2L2 may be involved in OA. To explore any potential participation of PMS2L2 in OA, synovial fluid was extracted from both OA patients and healthy controls (n = 62 each) and PMS2L2 expression of each sample determined by RT-qPCR. In addition, as miR-34a has a potential binding site on PMS2L2, hypothetical interactions between PMS2L2 and miR-34a in chondrocytes were analyzed by performing over-expression experiments. Furthermore, the role of PMS2L2 and miR-34a in the regulation of chondrocyte proliferation was analyzed using CCK-8 and BrdU assays. The results showed that PMS2L2 expression in OA patient synovial fluid was lower compared to that in control group fluid, and the extent of this reduction was related to disease stage. In in vitro studies, it was seen that endotoxin treatment of chondrocytes led to decreased PMS2L2 expression. It was found that PMS2L2 over-expression caused increased miR-34a expression in OA patient chondrocytes but not in cells from healthy controls. In contrast, miR-34a over-expression in either cell population did not affect PMS2L2 expression. Lastly, over-expression of both PMS2L2 and miR-34a led to inhibited chondrocyte proliferation. Of note, a combined over-expression of PMS2L2 and miR-34a resulted in stronger effects on proliferation compared to that from either single over-expression. Based on the findings that PMS2L2 is down-regulated during ongoing states of OA, and that changes in PMS2L2 expression can lead to increases in chondrocyte expression of miR-34a – resulting in inhibition of chondrocyte proliferation in OA. From these findings, one may conclude that finding means to regulate PMS2L2 could be a promising new target in the development of regimens for the treatment of OA.

MiR-29b inhibits TGF-β1-induced cell proliferation in articular chondrocytes

Transforming growth factor β1 (TGF-β1) is a known regulator of chondrocyte proliferation and promotes cartilage repair in osteoarthritis (OA). microRNA-29b-3p (miR-29b-3p) is downregulated by TGF-β1 and overexpressed in OA cartilage. However, the ability of miR-29b-3p to mediate the chondrocyte pro-proliferative effects of TGF-β1 is not yet understood. This current study aimed to investigate the effect of miR-29b-3p on TGF-β1-induced cell proliferation in murine articular chondrocytes. The stimulation of chondrocytes by TGF-β1 for 24 h resulted in the downregulation of miR-29b-3p expression. The ratio of G0/G1 phase cells decreased in response to TGF-β1 whereas the ratio of S phase cells was increased. Consistent with this observation, miR-29b-3p overexpression inhibited TGF-β1’s ability to promote the ratio of S phase cells and downregulate the ratio of G0/G1 phase cells. These findings suggest that the downregulation of miR-29b-3p is a likely requirement for TGF-β1-mediated proliferation of murine articular chondrocytes. Furthermore, implying that miR-29b-3p expression may be involved in reduced chondrocyte proliferation in OA.

Screening of the SHOX/PAR1 region using MLPA and miRNA expression profiling in a group of Egyptian children with non-syndromic short stature

BackgroundShort stature is defined as a body height below the third percentile, based on chronological age, or 2 standard deviations (SD) below the national height standard. The prevalence of short stature is around 2% of children worldwide. Several gene deficiencies have been associated with the etiology of short stature. The SHOX is an important candidate gene for short stature, as its haploinsufficiency underlies syndromic and non-syndromic short stature. Partial and complete duplications of SHOX have been reported in patients with short stature. Proper genetic diagnosis of these children allows for appropriate therapeutic approaches to be administered. Since copy number variation (CNV) is a possible mechanism of interhuman variability and pathogenic disease, the multiplex ligation-dependent probe amplification technique (MLPA) can be used as an initial screening technique. Cartilage tissue expresses specific microRNAs (miRNAs), which play an essential role in the regulation of chondrocyte proliferation and differentiation during growth plate development. We aimed to assess the SHOX/PAR1 region using CNV profiling for non-syndromic short stature in Egyptian children with and without growth hormone deficiency using the MLPA technique and expression profiling of miR-1, miR-15a, and miR-140 using quantitative real-time polymerase chain reaction (qRT-PCR) in a group of Egyptian children with non-syndromic short stature.ResultsOf the fifty cases included in this study, different CNVs were detected in ten children (20%), in/outside the SHOX region. Moreover, in children with short stature, the expression level of miRNA-140 was significantly different from that of healthy controls.ConclusionsThis is one of the first studies that have assessed CNVs in the SHOX/PAR1 region in a group of Egyptian children with short stature. MLPA analysis of SHOX/PAR1 identified different CNVs in children with non-syndromic short stature, suggesting that the MLPA should be used as an initial screening technique in short children, as proper genetic diagnosis of these children leads to implementation of the appropriate therapeutic approach. Alterations in the levels of miRNA-140 in children with short stature suggest that changes in the expression levels of this miRNA are associated with the pathogenesis of short stature.

Polygenic risk score for disability and insights into disability-related molecular mechanisms.

Late life disability is a highly devastating condition affecting 20% or more of persons aged 65 years and older in the USA; it is an important determinant of acute medical and long-term care costs which represent a growing burden on national economies. Disability is a multifactorial trait that contributes substantially to decline of health/wellbeing. Accordingly, gaining insights into the genetics of disability could help in identifying molecular mechanisms of this devastating condition and age-related processes contributing to a large fraction of specific geriatric conditions, concordantly with geroscience. We performed a genome-wide association study of disability in a sample of 24,068 subjects from five studies with 12,550 disabled individuals. We identified 30 promising disability-associated polymorphisms in 19 loci at p < 10-4; four of them attained suggestive significance, p < 10-5. In contrast, polygenic risk scores aggregating effects of minor alleles of independent SNPs that were adversely or beneficially associated with disability showed highly significant associations in meta-analysis, p = 3.13 × 10-45 and p = 5.60 × 10-23, respectively, and were replicated in each study. The analysis of genetic pathways, related diseases, and biological functions supported the connections of genes for the identified SNPs with disabling and age-related conditions primarily through oxidative/nitrosative stress, inflammatory response, and ciliary signaling. We identified musculoskeletal system development, maintenance, and regeneration as important components of gene functions. The beneficial and adverse gene sets may be differently implicated in the development of musculoskeletal-related disability with the beneficial set characterized, e.g., by regulation of chondrocyte proliferation and bone formation, and the adverse set by inflammation and bone loss.

Glucocorticoids Induces Apoptosis in Chondrocytes Through the Regulation of 11β-Hydroxysteroid Dehydrogenases (11β-HSDs)

Chondrocytes maintain the cartilaginous matrix and play an important role in endochondral ossification and cartilage repair. The regulation of chondrocyte proliferation and apoptosis is important in the pathology of cartilage and bone damage disorders. Recent studies have shown that glucocorticoids may induce apoptosis in chondrocytes. 11β-hydroxysteroid dehydrogenases (11β-HSDs) are microsomal enzymes that catalyze the interconversion of active 11-hydroxy-glucocorticoids and the inert 11-keto forms. We conducted an in vitro study to investigate the role of 11β-HSDs in apoptosis in femoral head chondrocytes. Our data showed that dexamethasone could induce chondrocyte apoptosis and cell viability reduction. Using Western blotting and real-time quantitative PCR, we observed that increased concentrations of dexamethasone increased the expression of 11β-HSD-1 and decreased the expression of 11β-HSD-2. Based on these results, we conclude that 11β-HSDs may be important for the regulation of glucocorticoid-induced apoptosis in chondrocytes. The present study demonstrates that glucocorticoids are able to induce both apoptosis in chondrocytes and a reduction in cell viability. Furthermore, 11β-HSDs may be important for the regulation of glucocorticoid-induced apoptosis in chondrocytes.

Evaluation of the chondroprotective action of N-acetylglucosamine in a rat experimental osteoarthritis model.

It has been demonstrated that oral administration of N-acetylglucosamine (GlcNAc) alleviates the symptoms of osteoarthritis (OA). The aim of the present study was to elucidate the molecular mechanisms for the chondroprotective action of GlcNAc in OA. Biomarkers for type II collagen degradation and synthesis were evaluated, as were histopathological changes, using a rat anterior cruciate ligament transection (ACLT)-induced OA model. Changes in the expression of genes in the cartilage were assessed via DNA microarray and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results indicated that ACLT induced histopathological changes of articular cartilage, whereas oral administration of GlcNAc (1,000 mg/kg/day for 28 days) significantly suppressed these changes. Additionally, GlcNAc significantly decreased levels of a type II collagen degradation marker in sera compared with that in the ACLT group, although there were no significant changes in the levels of a type II collagen synthesis marker. Furthermore, DNA microarray and reverse transcription-quantitative polymerase chain reaction results demonstrated that GlcNAc treatment downregulated the expression of periostin, which is likely involved in the degradation of cartilage, whereas GlcNAc upregulated the expression of lipocalin 2, which is involved in the regulation of chondrocyte proliferation and differentiation. In conclusion, the results of the present study suggest that GlcNAc is able to suppress the histopathological changes induced by OA and exhibits a chondroprotective action by inhibiting type II collagen degradation in the articular cartilage, possibly via modulation of the expression of inflammatory and chondroprotective molecules, including periostin and lipocalin 2.

Effects of miR-145 on the inhibition of chondrocyte proliferation and fibrosis by targeting TNFRSF11B in human osteoarthritis.

Osteoarthritis (OA) is a common cause of functional deterioration in older adults, and altered chondrogenesis is the most common pathophysiological process involved in the development of OA. MicroRNA‑145 (miR‑145) has been shown to regulate chondrocyte homeostasis. However, the function of miR‑145 in OA remains to be elucidated. In the present study, the expression levels of miR‑145 were examined in cartilage specimens from 25patients with knee OA using reverse transcription‑quantitative polymerase chain reaction analysis. The effects of miR‑145 on the proliferation and fibrosis of the C‑20/A4 and CH8 cell lines were also investigated using 3-(4,5-dimethylth-iazol-2-yl)-2,5-diphenyltetrazolium bromide and western blot assays invitro. The results revealed that the expression of miR-145 was decreased in the OA cartilage tissues, compared with normal cartilage tissues. The overexpression of miR‑145 by transfection of cells with miR‑145 mimics significantly inhibited C‑20/A4 and CH8 cell proliferation and fibrosis. Furthermore, tumor necrosis factor receptor superfamily, member 11b (TNFRSF11B) was identified as a direct target of miR‑145 in chondrocytes, which was confirmed using a dual‑luciferase reporter assay. The expression level of TNFRSF11B was markedly upregulated in the patients with OA, and the ectopic expression of miR‑145 was capable of suppressing the expression of TNFRSF11B. In addition, the knock down of TNFRSF11B using specific small interfering RNA also inhibited the proliferation and fibrosis of C‑20/A4 and CH8 cells invitro. These data provide the first evidence, to the best of our knowledge, to suggest the critical function of miR‑145 in regulating the expression of TNFRSF11B, which may have important implications on the regulation of chondrocyte proliferation and fibrosis in OA.

EZH1 and EZH2 promote skeletal growth by repressing inhibitors of chondrocyte proliferation and hypertrophy.

Histone methyltransferases EZH1 and EZH2 catalyse the trimethylation of histone H3 at lysine 27 (H3K27), which serves as an epigenetic signal for chromatin condensation and transcriptional repression. Genome-wide associated studies have implicated EZH2 in the control of height and mutations in EZH2 cause Weaver syndrome, which includes skeletal overgrowth. Here we show that the combined loss of Ezh1 and Ezh2 in chondrocytes severely impairs skeletal growth in mice. Both of the principal processes underlying growth plate chondrogenesis, chondrocyte proliferation and hypertrophy, are compromised. The decrease in chondrocyte proliferation is due in part to derepression of cyclin-dependent kinase inhibitors Ink4a/b, while ineffective chondrocyte hypertrophy is due to the suppression of IGF signalling by the increased expression of IGF-binding proteins. Collectively, our findings reveal a critical role for H3K27 methylation in the regulation of chondrocyte proliferation and hypertrophy in the growth plate, which are the central determinants of skeletal growth.

The PI3K/Akt signalling pathway may play an internal role related to abnormal condylar growth: a preliminary study

Developmental deformity of the mandible is one of the most common craniofacial malformations and is closely related to abnormal condylar growth. In this study, the role of PI3K/Akt signalling in the regulation of chondrocyte proliferation and hypertrophic differentiation in the condylar cartilage was studied. Immunohistochemical staining was used to investigate the expression of PI3K and p-Akt in the rat condyle cartilage. Rat condylar chondrocytes were cultured for the investigation of chondrocyte proliferation and hypertrophic differentiation when PI3K/Akt was inhibited. In addition, organ culture of the rat mandibular condyle was performed to evaluate the condyle cartilage growth while PI3K/Akt was inhibited. PI3K-positive cells and p-Akt-positive cells showing cytoplasmic staining were found to be present in the condylar cartilage. Reduced cell proliferation was observed in the culture of rat condylar chondrocytes when PI3K/Akt was inhibited; however, the hypertrophic differentiation level was increased. The proliferative zone thickness of condylar cartilage in the experimental group was less than that in the control group (P=0.00185), but the hypertrophic zone was greater than that in the control group (P=0.01048). PI3K/Akt signalling exerts opposite influences on chondrocyte proliferation and hypertrophic differentiation of the condylar cartilage, and these data suggest that PI3K/Akt is a potential intracellular regulation signal pathway in condylar cartilage development.

Regulation of chondrocyte proliferation through GIT1‐Rac1‐mediated ERK1/2 pathway by PDGF

There are many growth factors contributing to fracture healing after bone fractures. Platelet-derived growth factor (PDGF) released from platelets is a factor promoting cell division and proliferation, and first appears around the sites of fractures. Culture of chondrocytes in vitro are stimulated by PDGF to proliferation, its presence being upregulated in the extracellular matrix of cartilage; the main components include aggrecan and type II collagen. PDGF induces the expression of G the protein-coupled receptor kinase interacting protein 1 (GIT1), promoting Rac1 and ERK1/2 phosphorylation. Both knocking down GIT1 expression by siRNA and blocking phosphorylation of Rac1 inhibit this induced proliferation of chondrocyte. GIT1 and Rac1 control each other, having a synergistic effect on activation of the ERK1/2 pathway. The results suggest that PDGF regulates chondrocyte proliferation through activation of ERK1/2 pathway by upregulation of GIT1 expression and Rac1 phosphorylation.

Intraspecies disparity in growth rate is associated with differences in expression of local growth plate regulators

Disparities in longitudinal growth within a species can be partly explained by endocrinological differences. We hypothesized that regulatory networks acting locally in the growth plate may also be important. We tested this hypothesis by evaluating the IGF/IGFBP expression, the vitamin D pathway, and the PTHrP-Indian hedgehog (IHH) feedback loop in rib growth plates from 10- and 21-wk-old small- (Miniature Poodles, MP) and large-breed dogs (Great Danes, GD) using immunohistochemistry and quantitative (q)PCR. The rib growth plates of GD were 1.7 times thicker compared with those of MP, with larger proliferative (in absolute terms) and larger hypertrophic (in absolute and relative terms) zones. IGF/IGFBP gene expression profiling of the growth plates revealed decreased gene expression of igfbp2, -4, and -6 and an unaltered expression of igf-I and igf-II and their respective receptors in GD vs. MP. Immunohistochemistry and qPCR findings showed that the vitamin D pathway was more active in GD than in MP. Staining for 1α- and 24-hydroxylase was more abundant and intense in GD and the gene expressions of 1α-hydroxylase and the vitamin D receptor-driven 24-hydroxylase were six- and eightfold higher in GD vs. MP, respectively. Consistent with the immunohistochemistry findings, the expression of mRNA for components of the parathyroid hormone-related peptide (PTHrP)-IHH loop was different in GD compared with MP, with there being a relative threefold downregulation of Pthrp and a tenfold upregulation of Ihh in GD vs MP. These differences suggest that the effects of IHH in the regulation of chondrocyte proliferation and hypertrophy, both independently of PTHrP, can become more dominant during rapid growth rates. In conclusion, our data suggest that, in addition to modest endocrine differences, more pronounced changes in the expression of locally acting regulatory networks, such as the IGF system, vitamin D pathway, and PTHrP-IHH feedback loop are important contributors to within-species disparities in growth rates.

A splice‐site mutation leads to haploinsufficiency of EXT2 mRNA for a dominant trait in a large family with multiple osteochondromas

Multiple osteochondromas (MO) is an autosomal-dominant disorder and mutations in EXT1 and EXT2 account up to 78% of the cases studied, including missense, nonsense, frameshift, and splice-site mutations. EXT1 and EXT2 encode glycosyltransferases required for the synthesis of heparan sulfate (HS) chains. The molecular pathogenesis underlying these mutations is still largely unknown. A heterozygous c.1173 + 1G > T (EXT2) mutation was identified in a three-generation 34-member MO family and is present in all 19 affected members. The consequence of this mutation is exon 7 being spliced out, and the result is a shift in the codon-reading frame from position 360 (R360) of the amino acid sequence leading to a premature termination codon, and the mutant mRNA is degraded to an undetectable level. Interestingly, HS glycosaminoglycans were also undetectable in the cartilage cap of the tumors by immunostaining. Full penetrance of this mutation in all affected members ranging from 5 to 70 years of age suggests this primary defect in EXT2 mRNA level, in conjunction with other cellular changes such as enhanced heparanase expression, can produce profound effect on the synthesis of HS chains in cartilage, the consequence of which impacts on the regulation of chondrocyte proliferation and differentiation.

Signalling molecules and growth factors for tissue engineering of cartilage-what can we learn from the growth plate?

Modern tissue engineering concepts integrate cells, scaffolds, signalling molecules and growth factors. For the purposes of regenerative medicine, fetal development is of great interest because it is widely accepted that regeneration recapitulates in part developmental processes. In tissue engineering of cartilage the growth plate of the long bone represents an interesting, well-organized developmental structure with a spatial distribution of chondrocytes in different proliferation and differentiation stages, embedded in a scaffold of extracellular matrix components. The proliferation and differentiation of these chondrocytes is regulated by various hormonal and paracrine factors. Thus, members of the TGFbeta superfamily, the parathyroid hormone-related peptide-Indian hedgehog loop and a number of transcription factors, such as Sox and Runx, are involved in the regulation of chondrocyte proliferation and differentiation. Furthermore, adhesion molecules, homeobox genes, metalloproteinases and prostaglandins play a role in the complex regulation mechanisms. The present paper summarizes the morphological organization of the growth plate and provides a short but not exhaustive overview of the regulation of growth plate development, giving interesting insights for tissue engineering of cartilage.

Trps1, a regulator of chondrocyte proliferation and differentiation, interacts with the activator form of Gli3

Trps1, the gene mutated in human Tricho-Rhino-Phalangeal syndrome, represents an atypical member of the GATA-family of transcription factors. Here we show that Trps1 interacts with Indian hedgehog (Ihh)/Gli3 signaling and regulates chondrocyte differentiation and proliferation. We demonstrate that Trps1 specifically binds to the transactivation domain of Gli3 in vitro and in vivo, whereas the repressor form of Gli3 does not interact with Trps1. A domain of 185aa within Trps1, containing three predicted zinc fingers, is sufficient for interaction with Gli3. Using different mouse models we find that in distal chondrocytes Trps1 and the repressor activity of Gli3 are required to expand distal cells and locate the expression domain of Parathyroid hormone related peptide. In columnar proliferating chondrocytes Trps1 and Ihh/Gli3 have an activating function. The differentiation of columnar and hypertrophic chondrocytes is supported by Trps1 independent of Gli3. Trps1 seems thus to organize chondrocyte differentiation interacting with different subsets of co-factors in distinct cell types.

The Role of Cartilage Oligomeric Matrix Protein (COMP) in Skeletal Disease

Cartilage oligomeric matrix protein is a non-collagenous extracellular matrix protein expressed primarily in cartilage, ligament, and tendon. Cartilage oligomeric matrix protein has been studied extensively because mutations in the gene cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. Pseudoachondroplasia is a disproportionate dwarfing condition associated with joint abnormalities, while multiple epiphyseal dysplasia is less severe. Both of these skeletal dysplasias have a characteristic chondrocyte pathology that consists of intracellular retention of cartilage oligomeric matrix protein and other extracellular matrix proteins in an enlarged rough endoplasmic reticulum. This toxic intracellular retention of extracellular matrix proteins causes chondrocyte cell death thereby decreasing linear bone growth. Additionally, when cartilage oligomeric matrix protein and the other co-retained proteins are not exported to the extracellular matrix, the resulting matrix is abnormal and easily erodes with normal physical activity. Cartilage oligomeric matrix protein is also a marker for joint destruction associated osteoarthritis, rheumatoid arthritis, joint trauma, and intense activity. Serum cartilage oligomeric matrix protein levels are higher in aggressive cases of arthritis and levels are used to predict future disease progression. Recent studies have identified molecular functions of cartilage oligomeric matrix protein that may contribute to its role in skeletal disease. These molecular functions include: binding other ECM proteins, catalyzing polymerization of type II collagen fibrils, and regulation of chondrocyte proliferation. Here, we review cartilage oligomeric matrix protein's role in skeletal disease and potential molecular mechanisms.

Activating transcription factor-2 affects skeletal growth by modulating pRb gene expression

Endochondral ossification is the process of skeletal bone growth via the formation of a cartilage template that subsequently undergoes mineralization to form trabecular bone. Genetic mutations affecting the proliferation or differentiation of chondrocytes result in skeletal abnormalities. Activating transcription factor-2 (ATF-2) modulates expression of cell cycle regulatory genes in chondrocytes, and mutation of ATF-2 results in a dwarfed phenotype. Here we investigate the regulatory role that ATF-2 plays in expression of the pocket proteins, cell cycle regulators important in cellular proliferation and differentiation. The spatial and temporal pattern of pocket protein expression was identified in wild type and mutant growth plates. Expression of retinoblastoma (pRb) mRNA and protein were decreased in ATF-2 mutant primary chondrocytes. pRb mRNA expression was coordinated with chondrogenic differentiation and cell cycle exit in ATDC5 cells. Type X collagen immunohistochemistry was performed to visualize a delay in differentiation in response to loss of ATF-2 signaling. Chondrocyte proliferation was also affected by loss of ATF-2. These studies suggest pRb plays a role in chondrocyte proliferation, differentiation and growth plate development by modulating cell cycle progression. ATF-2 regulates expression of pRb within the developing growth plate, contributing to the skeletal phenotype of ATF-2 mutant mice through the regulation of chondrocyte proliferation and differentiation.

Dicer-dependent pathways regulate chondrocyte proliferation and differentiation

Small noncoding RNAs, microRNAs (miRNAs), bind to messenger RNAs through base pairing to suppress gene expression. Despite accumulating evidence that miRNAs play critical roles in various biological processes across diverse organisms, their roles in mammalian skeletal development have not been demonstrated. Here, we show that 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. Our results demonstrate the critical role of the Dicer-dependent pathway in the regulation of chondrocyte proliferation and differentiation during skeletal development.

Genetic variation in the PTH pathway and bone phenotypes in elderly women: Evaluation of PTH, PTHLH, PTHR1 and PTHR2 genes

Introduction Parathyroid hormone (PTH) is a key regulator of calcium metabolism. Parathyroid hormone-like hormone (PTHrP) contributes to skeletal development through regulation of chondrocyte proliferation and differentiation during early bone growth. Both PTH and PTHrP act through the same receptor (PTHR1). A second receptor, PTHR2, has been identified although its function is comparatively unknown. PTH hyper-secretion induces bone resorption, whereas intermittent injection of PTH increases bone mass. To explore the effects of genetic variation in the PTH pathway, we have analysed variations in PTH, PTHLH, PTHR1 and PTHR2 in relation to bone mass and fracture incidence in elderly women. Materials and methods This study includes 1044 elderly women, all 75 years old, from the Malmö Osteoporosis Prospective Risk Assessment study (OPRA). Single nucleotide polymorphisms (SNPs) from 4 genes and derived haplotypes in the PTH signaling pathway were analysed in 745–1005 women; 6 SNPs in the PTH gene and 3 SNPs each in the PTHLH, PTHR1 and PTHR2 genes were investigated in relation to BMD (assessed at baseline), fracture (434 prevalent fractures of all types over lifetime, self-reported and 174 incident fractures up to 7 years, X-ray verified) and serum PTH. Results and conclusion Individually, SNPs in the 4 loci did not show any significant association with BMD. Neither were PTHLH, PTHR1 and PTHR2 polymorphisms associated with fracture. Three of 5 common haplotypes, accounting for > 98% of alleles at the PTH locus, were identified as independent predictors of fracture. Haplotype 9 (19%) was suggestive of an association with fractures of any type sustained during lifetime ( p = 0.018), with carriers of one or more copies of the haplotype having the lowest incidence ( p = 0.006). Haplotypes 1 (13%) and 5 (37%) and 9 were suggestive of an association with fractures sustained between 50 and 75 years ( p = 0.02, p = 0.013 and p = 0.034). Carriers of haplotypes 1 and 5 were more likely to suffer a fracture (haplotype 1, p = 0.045; haplotype 5, p = 0.008). We conclude, that while further genotyping across the gene is recommended, in this cohort of elderly Swedish women, polymorphisms in PTH may contribute to the risk of fracture through mechanisms that are independent of BMD.

A functional polymorphism in the PTHR1 promoter region is associated with adult height and BMD measured at the femoral neck in a large cohort of young caucasian women

The parathyroid hormone type 1 receptor (PTHR1) mediates the actions of parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHRP). Interacting with this receptor, PTHRP contributes to skeletal development through the regulation of chondrocyte proliferation and differentiation. Recently, a tetranucleotide repeat-(AAAG)( n )-in the P3 promoter of the PTHR1 gene has been shown to have functional activity in vitro, and homozygosity for (AAAG)(6), or the 6/6 genotype, has been associated with greater adult height compared to the 5/5 genotype. In this study, we evaluated the association of (AAAG)( n ) with height and bone mineral density (BMD) measured at lumbar spine (LS) and femoral neck (FN) in a cohort of 677 young caucasian women 18-35 years of age. Genomic DNA was amplified and genotyped by comparison with sequenced controls following electrophoretic separation through high-resolution polyacrylamide gels. Allele frequencies for (AAAG)( n ) were: 76.8% (n=5); 20.9% (n=6); 1.8% (n=7); 0.18% (n=8); 0.27% (n=9); 0.08% (n=2), and there was no evidence for Hardy-Weinberg disequilibrium. Analysis of variance showed that subjects bearing one or two (AAAG)(6) alleles (6/X & 6/6) were significantly taller (165.7+/-0.5 cm) than the others (X/X, 164.5+/-0.3 cm; P=0.034). This association was significant after adjusting for multiple covariates-current age, age at menarche, physical activity, smoking status, and intakes of caffeine and calcium. Comparison of genotype groups for BMD was not significant at LS, but BMD was significantly higher at FN in the group with at least one (AAAG)(6) allele (adjusted means: 1.021+/-0.008 vs. 0.999+/-0.006 g/cm(2), P=0.032). In conclusion, our data show that subjects bearing one or two (AAAG)(6) alleles are taller than subjects without, reinforcing the notion that in vivo variation in promoter activity of the PTHR1 gene may be a relevant genetic influence on final adult height and BMD.