Twisted gastrulation modulates bone morphogenetic protein-induced collagen II and X expression in chondrocytes in vitro and in vivo

Twisted gastrulation (TSG) is an extracellular modulator of bone morphogenetic protein (BMP) activity and regulates dorsoventral axis formation in early Drosophila and Xenopus development. Studies on tsg-deficient mice also indicated a role of this protein in skeletal growth, but the mechanism of TSG activity in this process has not yet been investigated. Here we show for the first time by in situ hybridization and immunohistochemistry that TSG is strongly expressed in bovine and mouse growth plate cartilage as well as in fetal ribs, vertebral cartilage, and cartilage anlagen of the skull. Furthermore we provide evidence that TSG is directly involved in BMP-regulated chondrocyte differentiation and maturation. In vitro, TSG impaired the dose-dependent BMP-2 stimulation of collagen II and X expression in cultures of MC615 chondrocytes and primary mouse chondrocytes. In the presence of chordin, a BMP antagonist, the inhibitory effect of TSG was further enhanced. TSG also inhibited BMP-2-stimulated phosphorylation of Smad factors in chondrocytes, confirming the role of TSG as a modulator of BMP signaling. For analysis of TSG functions in cartilage development in vivo, the gene was overexpressed in transgenic mice under the control of the cartilage-specific Col2a1 promoter. As a result, Col10a1 expression was significantly reduced in the growth plates of transgenic embryos and newborns in comparison with wild type littermates as shown by in situ hybridization and by real time PCR analysis. The data suggest that TSG is an important modulator of BMP-regulated cartilage development and chondrocyte differentiation.     

Expression patterns and function of chromatin protein HMGB2 during mesenchymal stem cell differentiation

The superficial zone (SZ) of articular cartilage is critical in maintaining tissue function and homeostasis and represents the site of the earliest changes in osteoarthritis (OA). The expression of chromatin protein HMGB2 is restricted to the SZ, which contains cells expressing mesenchymal stem cell (MSC) markers. Age-related loss of HMGB2 and gene deletion are associated with reduced SZ cellularity and early onset OA. This study addressed HMGB2 expression patterns in MSC and its role during differentiation. HMGB2 was detected at higher levels in human MSC as compared with human articular chondrocytes, and its expression declined during chondrogenic differentiation of MSC. Lentiviral HMGB2 transduction of MSC suppressed chondrogenesis as reflected by an inhibition of Col2a1 and Col10a1 expression. Conversely, in bone marrow MSC from Hmgb2(-/-) mice, Col10a1 was more strongly expressed than in wild-type MSC. This is consistent with in vivo results from mouse growth plates showing that Hmgb2 is expressed in proliferating and prehypertrophic zones but not in hypertrophic cartilage where Col10a1 is strongly expressed. Osteogenesis was also accelerated in Hmgb2(-/-) MSC. The expression of Runx2, which plays a major role in late stage chondrocyte differentiation, was enhanced in Hmgb2(-/-) MSC, and HMGB2 negatively regulated the stimulatory effect of Wnt/β-catenin signaling on the Runx2 proximal promoter. These results demonstrate that HMGB2 expression is inversely correlated with the differentiation status of MSC and that HMGB2 suppresses chondrogenic differentiation. The age-related loss of HMGB2 in articular cartilage may represent a mechanism responsible for the decline in adult cartilage stem cell populations.     

Differential effects of osteogenic protein-1 (BMP-7) on gene expression of BMP and GDF family members during differentiation of the mouse MC615 chondrocyte cells

The mRNA expression patterns of several bone morphogenetic proteins (BMPs) and growth differentiation factors (GDFs) in long-term cultures of the clonal mouse chondrocyte cell line MC615 were examined. Distinct spatial and temporal patterns of expression of BMPs and GDFs were observed. The temporal orders of expression were correlated with those of several biochemical markers characteristic of chondrocytic cell differentiation. BMP-1, -2, -5, and -6 mRNA expression increased throughout the chondrogenic process and BMP-4 mRNA expression was not changed. GDF-1 and -3 mRNA expression increased throughout the chondrogenic process, and GDF-5, -6, -8, and -9 mRNA expressions were not changed. Effects of osteogenic protein-1 (OP-1, BMP-7) on the expression patterns of several other members of the BMP family and of the GDF family were also examined. OP-1 downregulated the BMP-1, -4, -5, and -6 mRNA expression by a maximal 3-, 5-, 2.5-, and 3-fold, respectively. The BMP-2 mRNA expression was not changed significantly by a low concentration of OP-1, but was increased at 200 ng/ml at day 7 of treatment. In contrast to the BMPs, OP-1 upregulated significantly the six GDF members examined (GDF-1, -3, -5, -6, -8, and -9) by three- to four-fold. Our findings demonstrate that OP-1 differentially regulates the mRNA expression of several related members of the BMP family and upregulates the mRNA expression of several members of the GDF family. The observations suggest that OP-1 action on cartilage differentiation involves a complex regulation of gene expression of several members of the BMP and the GDF family.     

软骨寡聚基质蛋白过表达对BMP-2诱导骨髓间充质干细胞分化的影响

目的：研究软骨寡聚基质蛋白（cartilage oligomeric matrix protein,COMP）过表达对BMP-2诱导骨髓间充质干细胞成骨及成软骨分化的影响。方法：BMP-2诱导骨髓间充质干细胞分化,通过脂质体转染含人COMP基因的质粒使骨髓间充质干细胞过表达COMP,采用实时定量PCR和Western blotting分析COMP基因过表达、成骨相关基因Ⅰ型胶原、RUNX2、骨钙蛋白以及成软骨相关基因Ⅱ型胶原、SOX9、蛋白聚糖、X型胶原的表达变化;通过茜素红染色观察成骨终末阶段矿化结节的生成情况,阿利新蓝染色观察细胞基质蛋白多糖的合成情况。结果：质粒转染后骨髓间充质干细胞COMP基因蛋白和mRNA表达水平显著提高（P<0.05）。COMP基因过表达后,成骨标记基因RUNX2、Ⅰ型胶原（Col1a1）mRNA水平均显著低于对照组（P<0.05）,RUNX2、骨钙蛋白（Osteocalcin）蛋白表达水平明显低于对照组（P<0.05）,而成软骨标记基因SOX9、蛋白聚糖（Aggrecan）mRNA水平均显著高于对照组（P<0.05）,SOX9、Ⅱ型胶原（Col2a1）蛋白表达均明显多于对照组（P<0.05）。细胞成骨茜素红染色弱于对照组,而阿利新蓝染色强于对照组。过表达组细胞X型胶原（Col10a1）基因表达显著低于对照组（P<0.05）,结论：骨髓间充质干细胞COMP基因过表达可抑制BMP-2诱导其成骨分化,促进骨髓间充质干细胞成软骨分化,并抑制软骨细胞的成熟肥大,为软骨组织工程研究提供新的方向。     

MSX2 stimulates chondrocyte maturation by controlling Ihh expression

Several studies indicated that a homeobox gene, Msx2, is implicated in regulation of skeletal development by controlling enchondral ossification as well as membranous ossification. However, the molecular basis by which Msx2 conducts chondrogenesis is currently unclear. In this study, we examined the role of Msx2 in chondrocyte differentiation using mouse primary chondrocytes and embryonic metatarsal explants. Treatment with BMP2 up-regulated the expression of Msx2 mRNA along with chondrocyte differentiation in murine primary chondrocytes. Overexpression of wild-type Msx2 stimulated calcification of primary chondrocytes in the presence of BMP2. We also found that constitutively active Msx2 (caMsx2) enhanced BMP2-dependent calcification more efficiently than wild-type Msx2. Consistently, caMsx2 overexpression up-regulated the expression of alkaline phosphatase and collagen type X induced by BMP2. Furthermore, organ culture experiments using mouse embryonic metatarsals indicated that caMsx2 clearly stimulated the maturation of chondrocytes into the prehypertrophic and hypertrophic stages in the presence of BMP2. In contrast, knockdown of Msx2 inhibited maturation of primary chondrocytes. The stimulatory effect of Msx2 on chondrocyte maturation was enhanced by overexpression of Smad1 and Smad4 but inhibited by Smad6, an inhibitory Smad for BMP2 signaling. These data suggest that Msx2 requires BMP2/Smad signaling for its chondrogenic action. In addition, caMsx2 overexpression induced Ihh (Indian hedgehog) expression in mouse primary chondrocytes. Importantly, treatment with cyclopamine, a specific inhibitor for hedgehogs, blocked Msx2-induced chondrogenesis. Collectively, our results indicated that Msx2 promotes the maturation of chondrocytes, at least in part, through up-regulating Ihh expression.     

Efficient differentiation of human iPSC‐derived mesenchymal stem cells to chondroprogenitor cells

Induced pluripotent stem cells (iPSC) hold tremendous potential for personalized cell‐based repair strategies to treat musculoskeletal disorders. To establish human iPSCs as a potential source of viable chondroprogenitors for articular cartilage repair, we assessed the in vitro chondrogenic potential of the pluripotent population versus an iPSC‐derived mesenchymal‐like progenitor population. We found the direct plating of undifferentiated iPSCs into high‐density micromass cultures in the presence of BMP‐2 promoted chondrogenic differentiation, however these conditions resulted in a mixed population of cells resembling the phenotype of articular cartilage, transient cartilage, and fibrocartilage. The progenitor cells derived from human iPSCs exhibited immunophenotypic features of mesenchymal stem cells (MSCs) and developed along multiple mesenchymal lineages, including osteoblasts, adipocytes, and chondrocytes in vitro. The data indicate the derivation of a mesenchymal stem cell population from human iPSCs is necessary to limit culture heterogeneity as well as chondrocyte maturation in the differentiated progeny. Moreover, as compared to pellet culture differentiation, BMP‐2 treatment of iPSC‐derived MSC‐like (iPSC–MSC) micromass cultures resulted in a phenotype more typical of articular chondrocytes, characterized by the enrichment of cartilage‐specific type II collagen (Col2a1), decreased expression of type I collagen (Col1a1) as well as lack of chondrocyte hypertrophy. These studies represent a first step toward identifying the most suitable iPSC progeny for developing cell‐based approaches to repair joint cartilage damage. J. Cell. Biochem. 114: 480–490, 2013. © 2012 Wiley Periodicals, Inc.     

Distinct functions of BMP4 and GDF5 in the regulation of chondrogenesis

Bone morphogenetic protein 4 (BMP4) and growth/differentiation factor 5 (GDF5) are closely related protein family members and regulate early cartilage patterning and differentiation. In this study, we compared the functional outcome of their actions systematically at various stages of chondrogenesis in mouse embryonic limb bud mesenchyme grown in micromass cultures. Overall, both growth factors enhanced cartilage growth and differentiation in these cultures. Uniquely, BMP4 not only accelerated the formation and maturation of cartilaginous nodules, but also induced internodular mesenchymal cells to express cartilage differentiation markers. On the other hand, GDF5 increased the number of prechondrogenic mesenchymal cell condensation and cartilaginous nodules, without altering the overall pattern of differentiation. In addition, GDF5 caused a more sustained elevated expression level of Sox9 relative to that associated with BMP4. BMP4 accelerated chondrocyte maturation throughout the cultures and sustained an elevated level of Col10 expression, whereas GDF5 caused a transient increase in Col10 expression. Taken together, we conclude that BMP4 is instructive to chondrogenesis and induces mesenchymal cells toward the chondrogenic lineage. Furthermore, BMP4 accelerates the progression of cartilage differentiation to maturation. GDF5 enhances cartilage formation by promoting chondroprogenitor cell aggregation, and amplifying the responses of cartilage differentiation markers. These differences may serve to fine-tune the normal cartilage differentiation program, and can be exploited for the molecular manipulation in biomimetics.     

Transgenic mice that express Cre recombinase in hypertrophic chondrocytes

In order to investigate the physiological control of hypertrophic chondrocytes which present the terminally differentiated form of chondrocytes, we generated a mouse line expressing the Cre recombinase under the control of the mouse type X collagen (Col10a1) promoter. In situ hybridization analysis demonstrated the expression of Col10a1-Cre transgene in hypertrophic chondrocytes of femur at postnatal day 2 (P2). In order to test the excision activity of the Cre recombinase, the Col10a1-Cre transgenic line was crossed with the mouse strain carrying the Smad4 conditional alleles (Smad4co/co) and the reporter line ROSA26. Multiple tissue PCR of Col10a1-Cre;Smad4co/+ mice revealed the restricted Cre activity in tissues containing hypertrophic chondrocytes. LacZ staining revealed that the Cre activity was observed in the cartilage primordia of ribs at E14.5 and only detected in the lower hypertrophic region of ribs at P1. These data suggest that the Col10a1-Cre mouse line described here could be used to achieve conditional gene targeting in hypertrophic chondrocytes.     

Distinct roles of BMP receptors Type IA and IB in osteo-/chondrogenic differentiation in mesenchymal progenitors (C3H10T1/2)

The functional roles of BMP type IA and IB receptors mediating differentiation into the osteogenic and chondrogenic lineage were investigated in the mesenchymal progenitor line C3H10T1/2 in vitro. The capacity of type IA and IB BMP receptors was assessed by the forced expression of the wild-type (wtBMPR-IA or IB) and of the kinase-deficient, dominant-negative form (dnBMPR-IA or -IB) in parental C3H10T1/2 progenitors as well as in C3H10T1/2 progenitors which recombinantly express BMP2 (C3H10T1/2-BMP2) or GDF5 (C3H10T1/2-GDF5). Consistent with the higher endogenous expression rate of BMPR-IA in comparison with BMPR-IB, BMPR-IA plays the dominant role in BMP2-mediated osteo-/chondrogenic development. BMPR-IB moderately influences osteogenic and hardly chondrogenic development. BMPR-IB seems to be unable to efficiently activate downstream signaling pathways upon forced expression. However, a mutation conferring constitutive activity to the BMPR-IB receptor indicates that this receptor possesses the capacity to activate downstream signaling cascades. These results suggest that in mesenchymal progenitors C3H10T1/2 BMPR-IA is responsible for the initiation of the osteogenic as well as chondrogenic development and that BMPR-IA and -IB receptor pathways are well separated in this mesenchymal progenitor line and may not substitute each other. In addition this indicates that type IB and IA BMP receptors may transmit different signals during the specification and differentiation of mesenchymal lineages.     

The Effects of Ce on the Proliferation, Osteogenic Differentiation and Mineralization Function of MC3T3-E1 Cells In Vitro

The effects of Ce on the proliferation, osteogenic differentiation and mineralization function of a murine preosteoblast cell line MC3T3-E1 in vitro were investigated at cell and molecular levels. The results showed that Ce promoted the proliferation, osteogenic differentiation and mineralization function of MC3T3-E1 cells at concentrations of 0.0001, 0.001, 0.01, 0.1 and 1???M, but turned to inhibit the proliferation, osteogenic differentiation and mineralization function at concentrations of 10, 100 and 1000???M. Ce displayed the up-regulation of Runx2, BMP2, ALP, BSP, Col I and OCN genes at concentrations of 0.0001 and 0.1???M; these genes were down-regulated in the MC3T3-E1 cells treated with 1000???M Ce. The expression of BMP2, Runx2 and OCN proteins was promoted by Ce at concentrations of 0.0001 and 0.1???M, but these proteins were down-regulated after 1000???M Ce treatment. The results suggest that Ce likely up-regulates or down-regulates the expression of Runx2, which subsequently up- or down-regulates OB marker genes Col I and BMP2 at early stages and ALP and OCN at later stages of differentiation, thus causing to promote or inhibit the proliferation, osteogenic differentiation and mineralization function of MC3T3-E1 cells.