Regulation Of Skeletal Development Research Articles

Regulation of Skeletal Development and Maintenance by Runx2 and Sp7

Regulation of Skeletal Development and Maintenance by Runx2 and Sp7

KLF5 promotes the ossification process of ligamentum flavum by transcriptionally activating CX43

BackgroundOssification of ligamentum flavum (OLF) is a prevalent degenerative spinal disease, typically causing severe neurological dysfunction. Kruppel-like factor 5 (KLF5) plays an essential role in the regulation of skeletal development. However, the mechanism KLF5 plays in OLF remains unclear, necessitating further investigative studies.MethodsqRT-PCR, immunofluorescent staining and western blot were used to measure the expression of KLF5. Alkaline Phosphatase (ALP) staining, Alizarin red staining (ARS), and the expression of Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), and osteocalcin (OCN) were used to evaluate the osteogenic differentiation. Luciferase activity assay and ChIP-PCR were performed to investigate the molecular mechanisms.ResultsKLF5 was significantly upregulated in OLF fibroblasts in contrast to normal ligamentum flavum (LF) fibroblasts. Silencing KLF5 diminished osteogenic markers and mineralized nodules, while its overexpression had the opposite effect, confirming KLF5’s role in promoting ossification. Moreover, KLF5 promotes the ossification of LF by activating the transcription of Connexin 43 (CX43), and overexpressing CX43 could reverse the suppressive impact of KLF5 knockdown on OLF fibroblasts’ osteogenesis.ConclusionKLF5 promotes the OLF by transcriptionally activating CX43. This finding contributes significantly to our understanding of OLF and may provide new therapeutic targets.

Sonic Hedgehog potentiates BMP9-induced osteogenic differentiation of mesenchymal stem cells

Bone morphogenetic protein 9 (BMP9) has remarkable potential to induce the differentiation of mesenchymal stem cells (MSCs) towards the osteoblastic lineage. Additionally, research suggests that certain growth factors have the ability to potentiate BMP9-induced osteogenic differentiation of MSCs. Sonic Hedgehog (Shh) plays an indispensable role in the regulation of skeletal development. The objective of this research was to assess the potential influence of Shh on BMP9-induced osteogenic differentiation of MSCs. Our findings indicated that Shh effectively enhanced BMP9-induced early and late osteogenic differentiation of MSCs, and increased BMP9-induced expression/transcriptional activity of osteogenesis-related transcription factors. Besides, it was observed that Shh promoted BMP9-induced ectopic bone formation of MSCs in vivo. Moreover, BMP9 was able to facilitate the repair of bone defects in rats, while Shh further accelerated this reparative process. Mechanistically, Shh enhanced the activation of the Smad1/5/8 signaling pathway which was induced by BMP9. Furthermore, GANT-61, an inhibitor of Gli1 and Gli2, attenuated the enhancing effect of Shh on BMP9-induced osteogenic differentiation of MSCs. Collectively, the co-administration of BMP9 and Shh may present a promising therapeutic approach for the treatment of fracture nonunion, delayed fracture healing, and bone defects.

GRK2, a novel regulator of skeletal development

STUDY OBJECTIVEDuring skeletal development, the cartilaginous growth plate (GP) allows for bone elongation, composed of chondrocytes organized into three histological zones: resting (RZ), proliferative (PZ), and hypertrophic (HZ). Resting chondrocytes proliferate, become prehypertrophic, then hypertrophic, to create room for bone deposition. The G‐protein coupled receptor (GPCR) Parathyroid Hormone Receptor‐1 (PTH1R) expressed on the chondrocytes of the prehypertrophic zone, decelerates the transition to hypertrophy. PTH1R signaling is regulated by GPCR kinase 2 (GRK2); GRK2 phosphorylates PTH1R leading to its internalization and signal termination. Recent studies showed that loss of function mutations in GRK2 result in skeletal deformities, and we demonstrated that GRK2 promotes chondrocyte hypertrophy (CH) in adult arthritic cartilage. However, the role of GRK2 in molecular signaling in GP chondrocytes has yet to be explored.HYPOTHESISWe hypothesized that GRK2 desensitizes PTH1R to promote CH in the GP.METHODSWe utilized the Cre‐Lox system to generate transgenic mice with inducible ‐conditional‐knockout of GRK2 (GRK2‐icKO) in chondrocytes. KO was initiated at postnatal day 3 (PN3) by Tamoxifen ip injections and mice were sacrificed at PN10 to examine the effects on early and late stages of skeletal development. We performed H&E staining to analyze histological changes in GP structure. To determine changes in molecular signaling, we used ISH and IF staining to quantify expression of GRK2, PTH1R, BrdU (proliferation marker), and ColX (CH marker). At the chondro‐osseous junction, we quantified apoptosis (TUNEL staining), bone deposition (Col1 and Runx2 IF), and matrix mineralization (Alizarin Red (AR) staining). We used in vitro experiments with primary chondrocyte culture to confirm in vivoresults. GRK2 knockdown (KD) was achieved with siRNA before inducing CH. Gene and protein expression of the same molecular markers were analyzed with qPCR and WB, and matrix mineralization quantified by AR.RESULTSGKR2‐icKO mice had: (1) Shorter bones (2) Retained PTH1R expression in the HZ, with reduced gene expression (Fig. 1A), as confirmed in vitro (Fig. 1D) (3) Longer PZ and more proliferative chondrocytes confirmed by BrdU, (4) shorter HZ and less hypertrophic chondrocytes, confirmed by ColX IF (Fig. 1B). (5) At the chondro‐osseous junction, GRK2‐icKO mice had reduced chondrocyte apoptosis, bone deposition, and matrix mineralization (Fig. 1C). In vitroexperiments demonstrated increased GRK2 expression with CH, where GRK2 KD reduced CH and matrix mineralization (Fig. 1D).CONCLUSIONSWe show a novel role of GRK2 in skeletal development as a major regulator of PTH1R in GP chondrocytes. GRK2‐icKO increases chondrocyte proliferation and reduces hypertrophy and apoptosis, leading to reduced bone deposition, matrix mineralization, and shorter bones. Altogether, this presents GRK2 as a novel therapeutic target for skeletal abnormalities.

Roles of Chondroitin Sulfate Proteoglycans as Regulators of Skeletal Development.

The extracellular matrix (ECM) is critically important for most cellular processes including differentiation, morphogenesis, growth, survival and regeneration. The interplay between cells and the ECM often involves bidirectional signaling between ECM components and small molecules, i.e., growth factors, morphogens, hormones, etc., that regulate critical life processes. The ECM provides biochemical and contextual information by binding, storing, and releasing the bioactive signaling molecules, and/or mechanical information that signals from the cell membrane integrins through the cytoskeleton to the nucleus, thereby influencing cell phenotypes. Using these dynamic, reciprocal processes, cells can also remodel and reshape the ECM by degrading and re-assembling it, thereby sculpting their environments. In this review, we summarize the role of chondroitin sulfate proteoglycans as regulators of cell and tissue development using the skeletal growth plate model, with an emphasis on use of naturally occurring, or created mutants to decipher the role of proteoglycan components in signaling paradigms.

Antisense oligonucleotides targeting Notch2 ameliorate the osteopenic phenotype in a mouse model of Hajdu-Cheney syndrome

Notch receptors play critical roles in cell-fate decisions and in the regulation of skeletal development and bone remodeling. Gain-of-function NOTCH2 mutations can cause Hajdu-Cheney syndrome, an untreatable disease characterized by osteoporosis and fractures, craniofacial developmental abnormalities, and acro-osteolysis. We have previously created a mouse model harboring a point 6955C→T mutation in the Notch2 locus upstream of the PEST domain, and we termed this model Notch2tm1.1Ecan Heterozygous Notch2tm1.1Ecan mutant mice exhibit severe cancellous and cortical bone osteopenia due to increased bone resorption. In this work, we demonstrate that the subcutaneous administration of Notch2 antisense oligonucleotides (ASO) down-regulates Notch2 and the Notch target genes Hes-related family basic helix-loop-helix transcription factor with YRPW motif 1 (Hey1), Hey2, and HeyL in skeletal tissue from Notch2tm1.1Ecan mice. Results of microcomputed tomography experiments indicated that the administration of Notch2 ASOs ameliorates the cancellous osteopenia of Notch2tm1.1Ecan mice, and bone histomorphometry analysis revealed decreased osteoclast numbers in Notch2 ASO-treated Notch2tm1.1Ecan mice. Notch2 ASOs decreased the induction of mRNA levels of TNF superfamily member 11 (Tnfsf11, encoding the osteoclastogenic protein RANKL) in cultured osteoblasts and osteocytes from Notch2tm1.1Ecan mice. Bone marrow-derived macrophage cultures from the Notch2tm1.1Ecan mice displayed enhanced osteoclastogenesis, which was suppressed by Notch2 ASOs. In conclusion, Notch2tm1.1Ecan mice exhibit cancellous bone osteopenia that can be ameliorated by systemic administration of Notch2 ASOs.

RAGE Signaling in Skeletal Biology.

The receptor for advanced glycation end products (RAGE) and several of its ligands have been implicated in the onset and progression of pathologies associated with aging, chronic inflammation, and cellular stress. In particular, the role of RAGE and its ligands in bone tissue during both physiological and pathological conditions has been investigated. However, the extent to which RAGE signaling regulates bone homeostasis and disease onset remains unclear. Further, RAGE effects in the different bone cells and whether these effects are cell-type specific is unknown. The objective of the current review is to describe the literature over RAGE signaling in skeletal biology as well as discuss the clinical potential of RAGE as a diagnostic and/or therapeutic target in bone disease. The role of RAGE and its ligands during skeletal homeostasis, tissue repair, and disease onset/progression is beginning to be uncovered. For example, detrimental effects of the RAGE ligands, advanced glycation end products (AGEs), have been identified for osteoblast viability/activity, while others have observed that low level AGE exposure stimulates osteoblast autophagy, which subsequently promotes viability and function. Similar findings have been reported with HMGB1, another RAGE ligand, in which high levels of the ligand are associated with osteoblast/osteocyte apoptosis, whereas low level/short-term administration stimulates osteoblast differentiation/bone formation and promotes fracture healing. Additionally, elevated levels of several RAGE ligands (AGEs, HMGB1, S100 proteins) induce osteoblast/osteocyte apoptosis and stimulate cytokine production, which is associated with increased osteoclast differentiation/activity. Conversely, direct RAGE-ligand exposure in osteoclasts may have inhibitory effects. These observations support a conclusion that elevated bone resorption observed in conditions of high circulating ligands and RAGE expression are due to actions on osteoblasts/osteocytes rather than direct actions on osteoclasts, although additional work is required to substantiate the observations. Recent studies have demonstrated that RAGE and its ligands play an important physiological role in the regulation of skeletal development, homeostasis, and repair/regeneration. Conversely, elevated levels of RAGE and its ligands are clearly related with various diseases associated with increased bone loss and fragility. However, despite the recent advancements in the field, many questions regarding RAGE and its ligands in skeletal biology remain unanswered.

Inhibin α-subunit inhibits BMP9-induced osteogenic differentiation through blocking BMP/Smad signal and activating NF-κB signal in mesenchymal stem cells.

Inhibin-α, a member of the transforming growth factor (TGF-β) superfamily, has been involved in bone turnover during the menopausal transition via endocrine effects, and it was previously reported that inhibins may antagonize the function of BMPs. Certainly, one of the most important functions of BMPs is to induce osteogenic differentiation. BMP9 as one of the most potent BMPs to induce osteogenic differentiation has gotten more and more attentions. Nonetheless, the effects of inhibin-α on osteogenesis remain unknown. Besides, mesenchymal stem cells (MSCs) with the ability to differentiate into multiple mesenchymal lineages, including osteoblasts, adipocyte, chondrocytes, and myoblasts in vitro, have become the promising seed cells for bone tissue engineering. Here, we investigated the role of inhibin-α on BMP9-induced osteogenic differentiation in MSCs and tried to discover the mechanism underlying this process. We found inhibin-α apparently reduced the classical osteogenic markers and the ectopic bone formation induced by BMP9. In addition, the ratio of OPG to RANKL is declined also in the presence of inhibin-α. For mechanism, we found that exogenous expression of inhibin-α inhibits BMP9-induced osteogenic differentiation through blocking BMP/Smad signal transduction and activating NF-κB signal which is repressed by BMP9. Thus, our findings indicated that inhibin-α has a negative effect on BMP9-induced osteogenic differentiation in MSCs, which may provide a novel insight into the regulation of skeletal development and new strategy for bone tissue engineering.

Loss of the Hematopoietic Stem Cell Factor GATA2 in the Osteogenic Lineage Impairs Trabecularization and Mechanical Strength of Bone.

The transcription factor GATA2 is required for expansion and differentiation of hematopoietic stem cells (HSCs). In mesenchymal stem cells (MSCs), GATA2 blocks adipogenesis, but its biological relevance and underlying genomic events are unknown. We report a dual function of GATA2 in bone homeostasis. GATA2 in MSCs binds near genes involved in skeletal system development and colocalizes with motifs for FOX and HOX transcription factors, known regulators of skeletal development. Ectopic GATA2 blocks osteoblastogenesis by interfering with SMAD1/5/8 activation. MSC-specific deletion of GATA2 in mice increases the numbers and differentiation capacity of bone-derived precursors, resulting in elevated bone formation. Surprisingly, MSC-specific GATA2 deficiency impairs the trabecularization and mechanical strength of bone, involving reduced MSC expression of the osteoclast inhibitor osteoprotegerin and increased osteoclast numbers. Thus, GATA2 affects bone turnover via MSC-autonomous and indirect effects. By regulating bone trabecularization, GATA2 expression in the osteogenic lineage may contribute to the anatomical and cellular microenvironment of the HSC niche required for hematopoiesis.

Recent advances in epigenetic regulation of skeletal development, homeostasis and disease

Recent advances in epigenetic regulation of skeletal development, homeostasis and disease

The Indispensable Role of Cyclin-Dependent Kinase 1 in Skeletal Development

Skeletal development is tightly regulated through the processes of chondrocyte proliferation and differentiation. Although the involvement of transcription and growth factors on the regulation of skeletal development has been extensively studied, the role of cell cycle regulatory proteins in this process remains elusive. To date, through cell-specific loss-of-function experiments in vivo, no cell cycle regulatory proteins have yet been conclusively shown to regulate skeletal development. Here, we demonstrate that cyclin-dependent kinase 1 (Cdk1) regulates skeletal development based on chondrocyte-specific loss-of-function experiments performed in a mouse model. Cdk1 is highly expressed in columnar proliferative chondrocytes and is greatly downregulated upon differentiation into hypertrophic chondrocytes. Cdk1 is essential for proper chondrocyte proliferation and deletion of Cdk1 resulted in accelerated differentiation of chondrocytes. In vitro and ex vivo analyses revealed that Cdk1 is an essential cell cycle regulatory protein for parathyroid hormone-related peptide (PTHrP) signaling pathway, which is critical to chondrocyte proliferation and differentiation. These results demonstrate that Cdk1 functions as a molecular switch from proliferation to hypertrophic differentiation of chondrocytes and thus is indispensable for skeletal development. Given the availability of inhibitors of Cdk1 activity, our results could provide insight for the treatment of diseases involving abnormal chondrocyte proliferation, such as osteoarthritis.

Sclerostin and dickkopf-1 are distinctly regulated by hyperthyroidism and hypothyroidism in mice

Introduction: The regulation of skeletal development through thyroid hormones (TH) remains poorly understood. Here, we tested the hypothesis that TH alter bone turnover by modulating dickkopf-1 (DKK1) and sclerostin (SOST), two inhibitors of the Wnt signaling pathway. Methods: We performed µCT, dynamic histomorphometry, and assessed bone remodeling markers and DKK1 and SOST levels in twelve-week old male hyperthyroid and hypothyroid C57BL/6 mice. The expression of osteoblast differentiation genes was evaluated using qPCR after treating murine osteoblasts with triiodothyronine (T3). Wnt activity was determined using a TCF/LEF promoter construct in MC3T3 cells exposed to T3. Results: Hyperthyroid mice showed a reduced trabecular (-54%, p < 0.001) and cortical bone density (-5%, p < 0.05) and cortical thickness (-15%, p < 0.001). In hypothyroid mice, trabecular bone density was higher (+26%, p < 0.001), but cortical bone parameters were unchanged. Histology, histomorphometry, and markers of bone remodeling (P1NP, CTX) indicated a high bone turnover in hyperthyroid and a low bone turnover in hypothyroid mice. Stimulation of osteoblasts with T3 led to an activation of Wnt signaling. Although serum DKK1 was decreased in hyperthyroid mice (-25%, p < 0.001) and increased in hypothyroid mice (+22%, p < 0.001), SOST was increased in both disease models. While SOST correlated positively with the bone density in euthyroid mice, this turned into a negative correlation when calculated across treatment groups. DKK1 did not correlate with bone density or bone remodeling parameters when calculated per treatment group. However, across all groups, DKK1 was positively correlated with BMD and strongly negatively correlated with bone formation and resorption parameters. SOST and DKK1 negatively correlated with each other. Conclusions: TH-induced changes in bone remodeling are distinctively associated with an altered Wnt activity in vitro and in vivo and a divergent regulation of DKK1 and SOST.

Sensorineural Hearing Loss in Hajdu-Cheney Syndrome

Sensorineural Hearing Loss in Hajdu-Cheney Syndrome Hajdu-Cheney Syndrome (HCS) is a rare connective tissue disorder which was first described in 1948 [1]. Less than 50 cases have been reported in the medical literature. The majority of cases are sporadic however an autosomal dominant inheritance pattern has been recognized [2-4]. The main characteristics of HCS include acroosteolysis, shortened terminal phalanges, short stature, joint hypermobility, as well as micrognathia. Cranial findings include open cranial suture lines, basilar invagination, posterior fossa abnormalities and hydrocephalus [2,4]. Conductive hearing loss has been reported in association with HCS [2,4,5], however sensorineural hearing loss has never been described in the literature. In particular the correlation between the course of the eighth cranial nerve and its mechanical stretching as a possible cause of sensorineural hearing loss remains unclear. Recent research has confirmed that heterogenous mutation in the Notch2 gene is the cause for Hajdu-Cheney syndrome [6]. The Notch pathway plays a key role in the regulation of skeletal development and bone remodeling as well as inner ear cellular differentiation.It is possible that alterations of intracochlear cell structure may lead to inner and outer hair cell changes that could in turn account for sensorineural hearing loss (SNHL).

Spatiotemporal expression and retinoic acid regulation of bone morphogenetic proteins 2, 4 and 16 in Senegalese sole

Summary Bone morphogenetic proteins (BMPs) are involved in various physiological processes from early life stages throughout adulthood. Specific characteristics of BMPs have been used to define different subfamilies and BMP2/4 subfamily (composed of BMP2 and BMP4) has been linked to osteogenesis and skeleton development. BMP16 was recently identified as a new member of the BMP2/4 subfamily and reported as a teleost fish-specific form. In this work, we collected a comprehensive set of ray-finned fish BMP2, BMP4 and BMP16 sequences and demonstrated, through its presence in Holostei, that BMP16 is not restricted to teleost fish genome. Comparative analysis of BMP2, BMP4 and BMP16 primary structures revealed that most of the residues required for protein stabilization, dimer formation, glycosylation and receptor binding are substantially conserved between the three proteins, suggesting that BMP16, BMP2 and BMP4 may share similar mechanisms of action. In contrast, comparative analysis of gene expression profiles during Senegalese sole development revealed differences in onset and extent of gene expression, indicating that BMP16, BMP2 and BMP4 may contribute to different developmental and physiological processes. High levels of transcripts in adult calcified tissues and the up-regulation of gene expression by retinoic acid, a known regulator of skeletal development, suggests that BMP16 shares with BMP2 and BMP4 a role in bone metabolism and skeletal development. This study provides new insights into the taxonomic distribution and the spatiotemporal expression of BMP16 gene, and suggests that it may share structural and functional similarities with other members of the BMP2/4 protein subfamily.

Multiple hereditary exostoses (MHE): elucidating the pathogenesis of a rare skeletal disorder through interdisciplinary research

An interdisciplinary and international group of clinicians and scientists gathered in Philadelphia, PA, to attend the fourth International Research Conference on Multiple Hereditary Exostoses (MHE), a rare and severe skeletal disorder. MHE is largely caused by autosomal dominant mutations in EXT1 or EXT2, genes encoding Golgi-associated glycosyltransferases responsible for heparan sulfate (HS) synthesis. HS chains are key constituents of cell surface- and extracellular matrix-associated proteoglycans, which are known regulators of skeletal development. MHE affected individuals are HS-deficient, can display skeletal growth retardation and deformities, and consistently develop benign, cartilage-capped bony outgrowths (termed exostoses or osteochondromas) near the growth plates of many skeletal elements. Nearly 2% of patients will have their exostoses progress to malignancy, becoming peripheral chondrosarcomas. Current treatments are limited to the surgical removal of symptomatic exostoses. No definitive treatments have been established to inhibit further formation and growth of exostoses, prevent transition to malignancy, or address other medical problems experienced by MHE patients, including chronic pain. Thus, the goals of the Conference were to assess our current understanding of MHE pathogenesis, identify key gaps in information, envision future therapeutic strategies and discuss ways to test and implement them. This report provides an assessment of the exciting and promising findings in MHE and related fields presented at the Conference and a discussion of the future MHE research directions. The Conference underlined the critical usefulness of gathering experts in several research fields to forge new alliances and identify cross-fertilization areas to benefit both basic and translational biomedical research on the skeleton.

Sensorineural Hearing Loss in Hajdu-Cheney Syndrome

Sensorineural Hearing Loss in Hajdu-Cheney Syndrome Hajdu-Cheney Syndrome (HCS) is a rare connective tissue disorder which was first described in 1948 [1]. Less than 50 cases have been reported in the medical literature. The majority of cases are sporadic however an autosomal dominant inheritance pattern has been recognized [2-4]. The main characteristics of HCS include acroosteolysis, shortened terminal phalanges, short stature, joint hypermobility, as well as micrognathia. Cranial findings include open cranial suture lines, basilar invagination, posterior fossa abnormalities and hydrocephalus [2,4]. Conductive hearing loss has been reported in association with HCS [2,4,5], however sensorineural hearing loss has never been described in the literature. In particular the correlation between the course of the eighth cranial nerve and its mechanical stretching as a possible cause of sensorineural hearing loss remains unclear. Recent research has confirmed that heterogenous mutation in the Notch2 gene is the cause for Hajdu-Cheney syndrome [6]. The Notch pathway plays a key role in the regulation of skeletal development and bone remodeling as well as inner ear cellular differentiation.It is possible that alterations of intracochlear cell structure may lead to inner and outer hair cell changes that could in turn account for sensorineural hearing loss (SNHL).

OP0050 Fine Mapping and Expression of a Locus Overlapping 3 Types of Inflammatory Arthritis

BackgroundIn a recent fine-mapping study using the Immunochip genotyping array, the RUNX1 region was associated with rheumatoid arthritis (RA p=5×10-10), juvenile idiopathic arthritis (JIA p=1×10-8) and psoriatic arthritis (PsA p=6×10-4)....

Abstract 1740: SATB2 localizes to mitotic microtubules and centrosome of cell cycles and regulates G1 phase cell cycle via proteosomal-dependent CDK2 in SKOV-3 ovarian cancer cells.

Abstract Though special AT-rich sequence-binding protein 2 (SATB2) is known as a transcriptional regulator of skeletal development and osteogenic differentiation, the underlying role of SATB2 still remains unclear. In the present study, we found that SATB2 localized to the mitotic microtubules, centrosome and midbodies in mitotic cells with α-tubulin. SATB2 knockdown by siRNA transfection reduced the cell viability, and increased the number of ethidium homodimer positive stained cells in SKOV3 ovarian cancer cells. Consistently, cell cycle analysis revealed that SATB2 knockdown accumulated G1 arrest and downregulated the cyclin-dependent kinase 2 (CDK2) expression by western blotting, while immunofluorescence assay revealed that CDK2 and SATB2 were co-localized in SKOV3 cells. Furthermore, proteosomal inhibitor MG132 treatment reversed the downregulation of CDK2 induced by SATB2 knockdown in SKOV3 cells. Overall, our findings suggest that SATB2 regulates the mitosis and G1 phase cell cycle via proteosomal ubiquitination in SKOV3 ovarian cancer cells. Citation Format: Eun Ah Sin, Eun J. Sohn, Ji Hoon Jung, Duckgue Lee, Bonglee Kim, Duck-Beom Jung, Ji-Hyun Kim, Hyo-Jeong Lee, Sung Hoon Kim. SATB2 localizes to mitotic microtubules and centrosome of cell cycles and regulates G1 phase cell cycle via proteosomal-dependent CDK2 in SKOV-3 ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1740. doi:10.1158/1538-7445.AM2013-1740

Deletion of SHP-2 in mesenchymal stem cells causes growth retardation, limb and chest deformity, and calvarial defects in mice.

SUMMARYIn mice, induced global disruption of the Ptpn11 gene, which encodes the SHP-2 tyrosine phosphatase, results in severe skeletal abnormalities. To understand the extent to which skeletal abnormalities can be attributed to perturbation of SHP-2 function in bone-forming osteoblasts and chondrocytes, we generated mice in which disruption of Ptpn11 is restricted to mesenchymal stem cells (MSCs) and their progeny, which include both cell types. MSC-lineage-specific SHP-2 knockout (MSC SHP-2 KO) mice exhibited postnatal growth retardation, limb and chest deformity, and calvarial defects. These skeletal abnormalities were associated with an absence of mature osteoblasts and massive chondrodysplasia with a vast increase in the number of terminally differentiated hypertrophic chondrocytes in affected bones. Activation of mitogen activated protein kinases (MAPKs) and protein kinase B (PKB; also known as AKT) was impaired in bone-forming cells of MSC SHP-2 KO mice, which provides an explanation for the skeletal defects that developed. These findings reveal a cell-autonomous role for SHP-2 in bone-forming cells in mice in the regulation of skeletal development. The results add to our understanding of the pathophysiology of skeletal abnormalities observed in humans with germline mutations in the PTPN11 gene (e.g. Noonan syndrome and LEOPARD syndrome).

Retinoic acid and the transcription factor MafB act together and differentially to regulate aggrecan and matrix metalloproteinase gene expression in neonatal chondrocytes

Vitamin A (VA) and its active form, retinoic acid (RA), are regulators of skeletal development and chondrogenesis. MafB, a transcription factor, has been identified as an important mediator in monocyte and osteoclast differentiation. However, the presence and function of MafB in chondrocytes is not clear. In this study, MafB gene expression was regulated by both the VA status of the mother (VA-marginal, adequate, and supplemented diets) and by direct oral supplementation of the neonates with VARA (VA mixed with 10% RA). Expression was highest in neonates of VA-supplemented versus VA-marginal dams (P < 0.05), and in VARA-treated versus placebo-treated neonates across all diet groups (P < 0.05). To examine cellular changes, primary chondrocytes derived from neonatal rat ribs were cultured in the presence of RA for up to 48 h. MafB mRNA exhibited a time- and dose-dependent increase in response to RA, while the induction of MafB mRNA was attenuated by BMS-493, a pan-RAR inverse agonist, implicating RAR signaling in the regulation of MafB. The genetic knockdown of MafB in chondrocytes using siRNA (MafB(SI) chondrocytes) abrogated the RA-induced increase in MafB expression. MafB(SI) chondrocytes expressed higher levels of aggrecan mRNA. Additionally, the increased matrix metalloproteinase (MMP)3 and MMP13 gene expression due to RA was attenuated in MafB(SI) chondrocytes, while total extracellular matrix staining was increased. These results support a role for MafB as a regulator of chondrocyte gene expression and matrix formation via control of aggrecan, MMP3 and MMP13 expression, and indicate an important role for RA in the regulation of MafB.