Jennifer H. Jonason

Post-translational Regulation of Runx2 in Bone and Cartilage

The Runx2 gene product is essential for mammalian bone development. In humans, Runx2 haploinsufficiency results in cleidocranial dysplasia, a skeletal disorder characterized by bone and dental abnormalities. At the molecular level, Runx2 acts as a transcription factor for genes expressed in hypertrophic chondrocytes and osteoblasts. Runx2 gene expression and protein function are regulated on multiple levels, including transcription, translation, and post-translational modification. Furthermore, Runx2 is involved in numerous protein-protein interactions, most of which either activate or repress transcription of target genes. In this review, we discuss expression of Runx2 during development as well as the post-translational regulation of Runx2 through modification by phosphorylation, ubiquitination, and acetylation.

Cartilage-specific β-catenin signaling regulates chondrocyte maturation, generation of ossification centers, and perichondrial bone formation during skeletal development.

The WNT/β‐catenin signaling pathway is a critical regulator of chondrocyte and osteoblast differentiation during multiple phases of cartilage and bone development. Although the importance of β‐catenin signaling during the process of endochondral bone development has been previously appreciated using a variety of genetic models that manipulate β‐catenin in skeletal progenitors and osteoblasts, genetic evidence demonstrating a specific role for β‐catenin in committed growth‐plate chondrocytes has been less robust. To identify the specific role of cartilage‐derived β‐catenin in regulating cartilage and bone development, we studied chondrocyte‐specific gain‐ and loss‐of‐function genetic mouse models using the tamoxifen‐inducible Col2CreERT2 transgene in combination with β‐cateninfx(exon3)/wt or β‐cateninfx/fx floxed alleles, respectively. From these genetic models and biochemical data, three significant and novel findings were uncovered. First, cartilage‐specific β‐catenin signaling promotes chondrocyte maturation, possibly involving a bone morphogenic protein 2 (BMP2)‐mediated mechanism. Second, cartilage‐specific β‐catenin facilitates primary and secondary ossification center formation via the induction of chondrocyte hypertrophy, possibly through enhanced matrix metalloproteinase (MMP) expression at sites of cartilage degradation, and potentially by enhancing Indian hedgehog (IHH) signaling activity to recruit vascular tissues. Finally, cartilage‐specific β‐catenin signaling promotes perichondrial bone formation possibly via a mechanism in which BMP2 and IHH paracrine signals synergize to accelerate perichondrial osteoblastic differentiation. The work presented here supports the concept that the cartilage‐derived β‐catenin signal is a central mediator for major events during endochondral bone formation, including chondrocyte maturation, primary and secondary ossification center development, vascularization, and perichondrial bone formation.

Axin2 controls bone remodeling through the β-catenin–BMP signaling pathway in adult mice

To investigate the role of Wnt–β-catenin signaling in bone remodeling, we analyzed the bone phenotype of female Axin2-lacZ knockout (KO) mice. We found that trabecular bone mass was significantly increased in 6- and 12-month-old Axin2 KO mice and that bone formation rates were also significantly increased in 6-month-old Axin2 KO mice compared with wild-type (WT) littermates. In vitro studies were performed using bone marrow stromal (BMS) cells isolated from 6-month-old WT and Axin2 KO mice. Osteoblast proliferation and differentiation were significantly increased and osteoclast formation was significantly reduced in Axin2 KO mice. Nuclear β-catenin protein levels were significantly increased in BMS cells derived from Axin2 KO mice. In vitro deletion of the β-catenin gene under Axin2 KO background significantly reversed the increased alkaline phosphatase activity and the expression of osteoblast marker genes observed in Axin2 KO BMS cells. We also found that mRNA expression of Bmp2 and Bmp4 and phosphorylated Smad1/5 protein levels were significantly increased in BMS cells derived from Axin2 KO mice. The chemical compound BIO, an inhibitor of glycogen synthase kinase 3β, was utilized for in vitro signaling studies in which upregulated Bmp2 and Bmp4 expression was measured in primary calvarial osteoblasts. Primary calvarial osteoblasts were isolated from Bmp2fx/fx;Bmp4fx/fx mice and infected with adenovirus-expressing Cre recombinase. BIO induced Osx, Col1, Alp and Oc mRNA expression in WT cells and these effects were significantly inhibited in Bmp2/4-deleted osteoblasts, suggesting that BIO-induced Osx and marker gene expression were Bmp2/4-dependent. We further demonstrated that BIO-induced osteoblast marker gene expression was significantly inhibited by Osx siRNA. Taken together, our findings demonstrate that Axin2 is a key negative regulator in bone remodeling in adult mice and regulates osteoblast differentiation through the β-catenin–BMP2/4–Osx signaling pathway in osteoblasts.

PTHrP prevents chondrocyte premature hypertrophy by inducing cyclin-D1-dependent Runx2 and Runx3 phosphorylation, ubiquitylation and proteasomal degradation

In chondrocytes, PTHrP maintains them in a proliferative state and prevents premature hypertrophy. The mechanism by which PTHrP does this is not fully understood. Both Runx2 and Runx3 are required for chondrocyte maturation. We recently demonstrated that cyclin D1 induces Runx2 protein phosphorylation and degradation. In the present studies, we tested the hypothesis that PTHrP regulates both Runx2 and Runx3 protein stability through cyclin D1. We analyzed the effects of cyclin D1 on Runx3 protein stability and function using COS cells, osteoprogenitor C3H10T1/2 cells and chondrogenic RCJ3.1C5.18 cells. We found that cyclin D1 induced Runx3 degradation in a dose-dependent manner and that both Myc-tagged Runx3 and endogenous Runx3 interact directly with CDK4 in COS and RCJ3.1C5.18 cells. A conserved CDK recognition site was identified in the C-terminal region of Runx3 by sequence analysis (residues 356-359). Pulse-chase experiments showed that the mutation of Runx3 at Ser356 to alanine (SA-Runx3) increased the half-life of Runx3. By contrast, the mutation at the same serine residue to glutamic acid (SE-Runx3) accelerated Runx3 degradation. In addition, SA-Runx3 was resistant to cyclin D1-induced degradation. GST-Runx3 was strongly phosphorylated by CDK4 in vitro. By contrast, CDK4 had no effect on the phosphorylation of SA-Runx3. Although both wild-type and SE-Runx3 were ubiquitylated, this was not the case for SA-Runx3. Runx3 degradation by cyclin D1 was completely blocked by the proteasome inhibitor PS1. In C3H10T1/2 cells, SA-Runx3 had a greater effect on reporter activity than SE-Runx3. The same was true for ALP activity in these cells. To investigate the role of cyclin D1 in chondrocyte proliferation and hypertrophy, we analyzed the growth plate morphology and expression of chondrocyte differentiation marker genes in Ccnd1-knockout mice. The proliferating and hypertrophic zones were significantly reduced and expression of chondrocyte differentiation marker genes and ALP activity were enhanced in 2-week-old Ccnd1-knockout mice. PTHrP significantly suppressed protein levels of both Runx2 and Runx3 in primary chondrocytes derived from wild-type mice. By contrast, the suppressive effect of PTHrP on Runx2 and Runx3 protein levels was completely abolished in primary chondrocytes derived from Ccnd1-knockout mice. Our findings demonstrate that the cell cycle proteins cyclin D1 and CDK4 induce Runx2 and Runx3 phosphorylation, ubiquitylation and proteasomal degradation. PTHrP suppresses Runx2 and Runx3 protein levels in chondrocytes through cyclin D1. These results suggest that PTHrP might prevent premature hypertrophy in chondrocytes, at least in part by inducing degradation of Runx2 and Runx3 in a cyclin-D1-dependent manner.

TAK1 Regulates Cartilage and Joint Development via the MAPK and BMP Signaling Pathways

The importance of canonical transforming growth factor β (TGF‐β) and bone morphogenetic protein (BMP) signaling during cartilage and joint development is well established, but the necessity for noncanonical (SMAD‐independent) signaling during these processes is largely unknown. TGF‐β activated kinase 1 (TAK1) is a MAP3K activated by TGF‐β, BMP, and other mitogen‐activated protein kinase (MAPK) signaling components. We set out to define the potential role for noncanonical, TAK1‐mediated signaling in cartilage and joint development via deletion of Tak1 in chondrocytes (Col2Cre;Tak1f/f) and the developing limb mesenchyme (Prx1Cre;Tak1f/f). Deletion of Tak1 in chondrocytes resulted in novel embryonic developmental cartilage defects including decreased chondrocyte proliferation, reduced proliferating chondrocyte survival, delayed onset of hypertrophy, reduced Mmp13 expression, and a failure to maintain interzone cells of the elbow joint, which were not observed previously in another Col2Cre;Tak1f/f model. Deletion of Tak1 in limb mesenchyme resulted in widespread joint fusions likely owing to the differentiation of interzone cells to the chondrocyte lineage. The Prx1Cre;Tak1f/f model also allowed us to identify novel columnar chondrocyte organization and terminal maturation defects owing to the interplay between chondrocytes and the surrounding mesenchyme. Furthermore, both our in vivo models and in vitro cell culture studies demonstrate that loss of Tak1 results in impaired activation of the downstream MAPK target p38, as well as diminished activation of the BMP/SMAD signaling pathway. Taken together, these data demonstrate that TAK1 is a critical regulator of both MAPK and BMP signaling and is necessary for proper cartilage and joint development.

Aging periosteal progenitor cells have reduced regenerative responsiveness to bone injury and to the anabolic actions of PTH 1-34 treatment.

A stabilized tibia fracture model was used in young (8-week old) and aged (1-year old) mice to define the relative bone regenerative potential and the relative responsiveness of the periosteal progenitor population with aging and PTH 1-34 (PTH) systemic therapy. Bone regeneration was assessed through gene expressions, radiographic imaging, histology/histomorphometry, and biomechanical testing. Radiographs and microCT showed increased calcified callus tissue and enhanced bone healing in young compared to aged mice. A key mechanism involved reduced proliferation, expansion, and differentiation of periosteal progenitor cell populations in aged mice. The experiments showed that PTH increased calcified callus tissue and torsional strength with a greater response in young mice. Histology and quantitative histomorphometry confirmed that PTH increased callus tissue area due primarily to an increase in bone formation, since minimal changes in cartilage and mesenchyme tissue area occurred. Periosteum examined at 3, 5, and 7 days showed that PTH increased cyclin D1 expression, the total number of cells in the periosteum, and width of the periosteal regenerative tissue. Gene expression showed that aging delayed differentiation of both bone and cartilage tissues during fracture healing. PTH resulted in sustained Col10a1 expression consistent with delayed chondrocyte maturation, but otherwise minimally altered cartilage gene expression. In contrast, PTH 1-34 stimulated expression of Runx2 and Osterix, but resulted in reduced Osteocalcin. β-Catenin staining was present in mesenchymal chondroprogenitors and chondrocytes in early fracture healing, but was most intense in osteoblastic cells at later times. PTH increased active β-catenin staining in the osteoblast populations of both young and aged mice, but had a lesser effect in cartilage. Altogether the findings show that reduced fracture healing in aging involves decreased proliferation and differentiation of stem cells lining the bone surface. While PTH 1-34 enhances the proliferation and expansion of the periosteal stem cell population and accelerates bone formation and fracture healing, the effects are proportionately reduced in aged mice compared to young mice. β-Catenin is induced by PTH in early and late fracture healing and is a potential target of PTH 1-34 effects.

Aberrant Hypertrophy in Smad3-Deficient Murine Chondrocytes Is Rescued by Restoring Transforming Growth Factor β-Activated Kinase 1/ Activating Transcription Factor 2 Signaling: A Potential Clinical Implication for Osteoarthritis

OBJECTIVE To investigate the biologic significance of Smad3 in the progression of osteoarthritis (OA), the crosstalk between Smad3 and activating transcription factor 2 (ATF-2) in the transforming growth factor beta (TGFbeta) signaling pathway, and the effects of ATF-2 overexpression and p38 activation in chondrocyte differentiation. METHODS Joint disease in Smad3-knockout (Smad3(-/-)) mice was examined by microfocal computed tomography and histologic analysis. Numerous in vitro methods including immunostaining, real-time polymerase chain reaction, Western blotting, an ATF-2 DNA-binding assay, and a p38 kinase activity assay were used to study the various signaling responses and protein interactions underlying the altered chondrocyte phenotype in Smad3(-/-) mice. RESULTS In Smad3(-/-) mice, an end-stage OA phenotype gradually developed. TGFbeta-activated kinase 1 (TAK1)/ATF-2 signaling was disrupted in Smad3(-/-) mouse chondrocytes at the level of p38 MAP kinase (MAPK) activation, resulting in reduced ATF-2 phosphorylation and transcriptional activity. Reintroduction of Smad3 into Smad3(-/-) cells restored the normal p38 response to TGFbeta. Phosphorylated p38 formed a complex with Smad3 by binding to a portion of Smad3 containing both the MAD homology 1 and linker domains. Additionally, Smad3 inhibited the dephosphorylation of p38 by MAPK phosphatase 1 (MKP-1). Both ATF-2 overexpression and p38 activation repressed type X collagen expression in wild-type and Smad3(-/-) chondrocytes. P38 was detected in articular cartilage and perichondrium; articular and sternal chondrocytes expressed p38 isoforms alpha, beta, and gamma, but not delta. CONCLUSION Smad3 is involved in both the onset and progression of OA. Loss of Smad3 abrogates TAK1/ATF-2 signaling, most likely by disrupting the Smad3-phosphorylated p38 complex, thereby promoting p38 dephosphorylation and inactivation by MKP-1. ATF-2 and p38 activation inhibit chondrocyte hypertrophy. Modulation of p38 isoform activity may provide a new therapeutic approach for OA.

TAK1 regulates SOX9 expression in chondrocytes and is essential for postnatal development of the growth plate and articular cartilages

Summary TAK1 is a MAP3K that mediates non-canonical TGF-&bgr; and BMP signaling. During the embryonic period, TAK1 is essential for cartilage and joint development as deletion of Tak1 in chondro-osteo progenitor cells leads to severe chondrodysplasia with defects in both chondrocyte proliferation and maturation. We have investigated the role of TAK1 in committed chondrocytes during early postnatal development. Using the Col2a1-CreERT2; Tak1f/f mouse model, we induced deletion of Tak1 at postnatal day 7 and characterized the skeletal phenotypes of these mice at 1 and 3 months of age. Mice with chondrocyte-specific Tak1 deletion exhibited severe growth retardation and reduced proteoglycan and type II collagen content in the extracellular matrix of the articular cartilage. We found reduced Col2a1 and Acan expression, but increased Mmp13 and Adamts5 expression, in Tak1-deficient chondrocytes along with reduced expression of the SOX trio of transcription factors, SOX9, SOX5 and SOX6. In vitro, BMP2 stimulated Sox9 gene expression and Sox9 promoter activity. These effects were reduced; however, following Tak1 deletion or treatment with a TAK1 kinase inhibitor. TAK1 affects both canonical and non-canonical BMP signal transduction and we found that both of these pathways contribute to BMP2-mediated Sox9 promoter activation. Additionally, we found that ATF2 directly binds the Sox9 promoter in response to BMP signaling and that this effect is dependent upon TAK1 kinase activity. These novel findings establish that TAK1 contributes to BMP2-mediated Sox9 gene expression and is essential for the postnatal development of normal growth plate and articular cartilages.

EP1(-/-) mice have enhanced osteoblast differentiation and accelerated fracture repair.

As a downstream product of cyclooxygenase 2 (COX‐2), prostaglandin E2 (PGE2) plays a crucial role in the regulation of bone formation. It has four different receptor subtypes (EP1 through EP4), each of which exerts different effects in bone. EP2 and EP4 induce bone formation through the protein kinase A (PKA) pathway, whereas EP3 inhibits bone formation in vitro. However, the effect of EP1 receptor signaling during bone formation remains unclear. Closed, stabilized femoral fractures were created in mice with EP1 receptor loss of function at 10 weeks of age. Healing was evaluated by radiographic imaging, histology, gene expression studies, micro–computed tomographic (µCT), and biomechanical measures. EP1−/− mouse fractures have increased formation of cartilage, increased fracture callus, and more rapid completion of endochondral ossification. The fractures heal faster and with earlier fracture callus mineralization with an altered expression of genes involved in bone repair and remodeling. Fractures in EP1−/− mice also had an earlier appearance of tartrate‐resistant acid phosphatase (TRAcP)–positive osteoclasts, accelerated bone remodeling, and an earlier return to normal bone morphometry. EP1−/− mesenchymal progenitor cells isolated from bone marrow have higher osteoblast differentiation capacity and accelerated bone nodule formation and mineralization in vitro. Loss of the EP1 receptor did not affect EP2 or EP4 signaling, suggesting that EP1 and its downstream signaling targets directly regulate fracture healing. We show that unlike the PGE2 receptors EP2 and EP4, the EP1 receptor is a negative regulator that acts at multiple stages of the fracture healing process. Inhibition of EP1 signaling is a potential means to enhance fracture healing.

Isolation and culture of neonatal mouse calvarial osteoblasts.

This chapter describes the isolation and culture of neonatal mouse calvarial osteoblasts. This primary cell population is obtained by sequential enzymatic digestion of the calvarial bone matrix and is capable of differentiating in vitro into mature osteoblasts that deposit a collagen extracellular matrix and form mineralized bone nodules. Maturation of the cultures can be monitored by gene expression analyses and staining for the presence of alkaline phosphatase or matrix mineralization. This culture system, therefore, provides a powerful model to test how various experimental conditions, such as the manipulation of gene expression, may affect osteoblast maturation and/or function.