Takayuki Furumatsu

Stage-Specific Secretion of HMGB1 in Cartilage Regulates Endochondral Ossification

ABSTRACT High mobility group box 1 protein (HMGB1) is a chromatin protein that has a dual function as a nuclear factor and as an extracellular factor. Extracellular HMGB1 released by damaged cells acts as a chemoattractant, as well as a proinflammatory cytokine, suggesting that HMGB1 is tightly connected to the process of tissue organization. However, the role of HMGB1 in bone and cartilage that undergo remodeling during embryogenesis, tissue repair, and disease is largely unknown. We show here that the stage-specific secretion of HMGB1 in cartilage regulates endochondral ossification. We analyzed the skeletal development of Hmgb1−/− mice during embryogenesis and found that endochondral ossification is significantly impaired due to the delay of cartilage invasion by osteoclasts, osteoblasts, and blood vessels. Immunohistochemical analysis revealed that HMGB1 protein accumulated in the cytosol of hypertrophic chondrocytes at growth plates, and its extracellular release from the chondrocytes was verified by organ culture. Furthermore, we demonstrated that the chondrocyte-secreted HMGB1 functions as a chemoattractant for osteoclasts and osteoblasts, as well as for endothelial cells, further supporting the conclusion that Hmgb1−/− mice are defective in cell invasion. Collectively, these findings suggest that HMGB1 released from differentiating chondrocytes acts, at least in part, as a regulator of endochondral ossification during osteogenesis.

Regulation of mechanical stress-induced MMP-13 and ADAMTS-5 expression by RUNX-2 transcriptional factor in SW1353 chondrocyte-like cells

OBJECTIVE To investigate the mechanism of mechanical stress-induced expression and regulation of aggrecanases and examine the role of runt-related transcription factor 2 (RUNX-2) in chondrocyte-like cells. METHODS SW1353 cells were seeded onto stretch chambers at a concentration of 5×10⁴ cells/chamber, and a uni-axial cyclic tensile strain (CTS) (0.5 Hz, 10% stretch) was applied for 30 min. Total RNA was extracted, reverse transcribed, and analyzed by polymerase chain reaction (PCR) and real-time PCR. RUNX-2 overexpression and small interfering RNA (siRNA) targeting RUNX-2 were used to investigate the role of RUNX-2 in CTS-induced gene expression. The involvement of diverse mitogen-activated protein kinase (MAPK) pathways in the activation of RUNX-2, MMP-13 and ADAMTS-5 during CTS was examined by Western blotting. RESULTS CTS induced expression of RUNX-2, MMP-13, ADAMTS-4, -5, and -9. Overexpression of RUNX-2 up-regulated expression of MMP-13 and ADAMTS-5, whereas RUNX-2 siRNA resulted in significant down-regulation of mechanically-induced MMP-13 and ADAMTS-5 expression. CTS induced activation of p38 MAPK, and CTS induction of RUNX-2, MMP-13 and ADAMTS-5 mRNA was down-regulated by the selective p38 MAPK inhibitor SB203580 but not by the p44/42 MAPK inhibitor U0126, or the JNK MAPK inhibitor JNK inhibitor II. CONCLUSIONS RUNX-2 might have a role as a key downstream mediator of p38s ability to regulate mechanical stress-induced MMP-13 and ADAMTS-5 expression.

Smad3 activates the Sox9-dependent transcription on chromatin

Transforming growth factor (TGF)-beta has an essential role for the Sry-type high-mobility-group box (Sox)-regulated chondrogenesis. Chondrogenic differentiation is also controlled by chromatin-mediated transcription. We have previously reported that TGF-beta-regulated Smad3 induces chondrogenesis through the activation of Sox9-dependent transcription. However, the cross-talk between TGF-beta signal and Sox9 on chromatin-mediated transcription has not been elucidated. In the present study, we investigated the activity of Smad3, Sox9, and coactivator p300 using an in vitro chromatin assembly model. Luciferase reporter assays revealed that Smad3 stimulated the Sox9-mediated transcription in a TGF-beta-dependent manner. Recombinant Sox9 associated with phosphorylated Smad3/4 and recognized the enhancer region of type II collagen gene. In vitro transcription and S1 nuclease assays showed that Smad3 and p300 cooperatively activated the Sox9-dependent transcription on chromatin template. The combination treatment of phosphorylated Smad3, Sox9, and p300 were necessary for the activation of chromatin-mediated transcription. These findings suggest that TGF-beta signal Smad3 plays a key role for chromatin remodeling to induce chondrogenesis via its association with Sox9.

Scleraxis and E47 cooperatively regulate the Sox9-dependent transcription.

During musculoskeletal development, Sry-type HMG box 9 (Sox9) has a crucial role in mesenchymal condensation and chondrogenesis. On the other hand, a tissue-specific basic helix-loop-helix (bHLH) transcription factor Scleraxis (Scx) regulates the differentiation of tendon and ligament progenitors. Whereas these two transcription factors cooperatively participate in the determination of cellular lineages, the precise interaction between Sox9 and Scx remains unclear. We have previously demonstrated that the Sox9-dependent transcription is synergistically activated by several Sox9-associating molecules, such as p300 and Smad3, on chromatin. In this study, we investigated the function of Scx in the Sox9-dependent transcription. The expression of alpha1(II) collagen (Col2a1) gene was stimulated by an appropriate transduction of Sox9 and Scx. Scx and its partner E47, which dimerizes with other bHLH proteins, cooperatively enhanced the Sox9-dependent transcription in luciferase reporter assays. Coactivator p300 synergistically increased the activity of Sox9-regulated reporter gene, which contains promoter and enhancer regions of Col2a1, in the presence of Scx and E47. Immunoprecipitation analyses revealed that Scx and E47 formed a transcriptional complex with Sox9 and p300. Scx/E47 heterodimer also associated with a conserved E-box sequence (CAGGTG) in the Col2a1 promoter on chromatin. These findings suggest that Scx and E47 might modulate the primary chondrogenesis by associating with the Sox9-related transcriptional complex, and by binding to the conserved E-box on Col2a1 promoter.

Human chondrosarcoma secretes vascular endothelial growth factor to induce tumor angiogenesis and stores basic fibroblast growth factor for regulation of its own growth.

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are well‐known factors that induce neovascularization in many tumors. The molecular mechanisms that regulate tumor angiogenesis in human chondrosarcoma are not clear. We assessed in this work the angiogenic activities of a human chondrosarcoma cell line (OUMS‐27) in vivo and determined the efficacies of angiogenic factors derived from OUMS‐27 cells on human umbilical vein endothelial cells (HUVECs) in vitro. Tumor xenografts induced an increase in the formation of neovessels, but the distributions of Ki‐67 antigen, VEGF and bFGF were unaffected. We also demonstrated that OUMS‐27 cells secreted VEGF165 into the culture medium and that it was the maximal angiogenic factor to stimulate endothelial proliferation and migration in chondrosarcoma. Anti‐VEGF antibodies induced an approximately 70% inhibition of these responses of HUVECs, but did not have any effect on OUMS‐27 cells. Anti‐bFGF antibodies suppressed not only the activities of HUVECs but also the growth of tumor cells in vitro. We indicate that angiogenesis is principally elicited by VEGF165 and that tumorigenesis is mainly regulated by bFGF stored in the extracellular matrix of OUMS‐27 cells. The present study may offer the availability of combination therapies for inhibition of VEGF and bFGF action on vascular endothelial cells and chondrosarcoma cells, respectively.

Anterior cruciate ligament-derived cells have high chondrogenic potential

Anterior cruciate ligament (ACL)-derived cells have a character different from medial collateral ligament (MCL)-derived cells. However, the critical difference between ACL and MCL is still unclear in their healing potential and cellular response. The objective of this study was to investigate the mesenchymal differentiation property of each ligament-derived cell. Both ligament-derived cells differentiated into adipogenic, osteogenic, and chondrogenic lineages. In chondrogenesis, ACL-derived cells had the higher chondrogenic property than MCL-derived cells. The chondrogenic marker genes, Sox9 and alpha1(II) collagen (Col2a1), were induced faster in ACL-derived pellets than in MCL-derived pellets. Sox9 expression preceded the increase of Col2a1 in both pellet-cultured cells. However, the expression level of Sox9 and a ligament/tendon transcription factor Scleraxis did not parallel the increase of Col2a1 expression along with chondrogenic induction. The present study demonstrates that the balance between Sox9 and Scleraxis have an important role in the chondrogenic differentiation of ligament-derived cells.

Histone deacetylase inhibitors suppress mechanical stress-induced expression of RUNX-2 and ADAMTS-5 through the inhibition of the MAPK signaling pathway in cultured human chondrocytes.

OBJECTIVE To investigate the inhibitory effects and the regulatory mechanisms of histone deacetylase (HDAC) inhibitors on mechanical stress-induced gene expression of runt-related transcription factor (RUNX)-2 and adisintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-5 in human chondrocytes. METHODS Human chondrocytes were seeded in stretch chambers at a concentration of 5 × 10(4)cells/chamber. Cells were pre-incubated with or without HDAC inhibitors (MS-275 or trichostatin A; TSA) for 12h, followed by uniaxial cyclic tensile strain (CTS) (0.5Hz, 10% elongation), which was applied for 30 min using the ST-140-10 system (STREX, Osaka, Japan). Total RNA was extracted and the expression of RUNX-2, ADAMTS-5, matrix metalloproteinase (MMP)-3, and MMP-13 at the mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry, respectively. The activation of diverse mitogen-activated protein kinase (MAPK) pathways with or without HDAC inhibitors during CTS was examined by western blotting. RESULTS HDAC inhibitors (TSA: 10 nM, MS-275: 100 nM) suppressed CTS-induced expression of RUNX-2, ADAMTS-5, and MMP-3 at both the mRNA and protein levels within 1h. CTS-induced activation of p38 MAPK (p38), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) MAPKs was downregulated by both HDAC inhibitors. CONCLUSION The CTS-induced expression of RUNX-2 and ADAMTS-5 was suppressed by HDAC inhibitors via the inhibition of the MAPK pathway activation in human chondrocytes. The results of the current study suggested a novel therapeutic role for HDAC inhibitors against degenerative joint disease such as osteoarthritis.

GDF-5/7 and bFGF activate integrin α2-mediated cellular migration in rabbit ligament fibroblasts

Cellular activities responding to growth factors are important in ligament healing. The anterior cruciate ligament (ACL) has poor healing potential compared to the medial collateral ligament (MCL). To assess the differences, we investigated the proliferation, migration, adhesion, and matrix synthesis responding to growth factors in rabbit ACL and MCL fibroblasts. ACL cell proliferation to basic fibroblast growth factor (bFGF), bone morphogenetic protein‐2, growth and differentiation factor (GDF)‐5, and GDF‐7 treatment was similar to that of MCL cells. GDF‐5 enhanced Col1a1 expression in ACL and MCL fibroblasts up to 4.7‐ and 17‐fold levels of control, respectively. MCL fibroblasts showed stronger migration activities in response to bFGF and GDF‐5 than ACL cells. GDF‐5/7 and bFGF also changed the stress fiber formation and cellular adhesion by modulating the distribution of integrin α2. Functional blocking analyses using anti‐integrin α2 antibodies revealed that cellular migration responding to growth factors depended on the integrin α2‐mediated adhesion on type I collagen. The expression of integrin α2 was also increased by growth factors in both cells. Our results demonstrate that GDF‐5/7 and bFGF stimulate cellular migration by modulating integrin α2 expression and integrin α2‐dependent adhesion, especially in MCL fibroblasts. These findings suggest that the different healing potential between ACL and MCL may be caused by different cellular behavior in the integrin α2‐mediated cellular migration in response to growth factors.

Mechanical stretch stimulates integrin αVβ3-mediated collagen expression in human anterior cruciate ligament cells

Biomechanical stimuli have fundamental roles in the maintenance and remodeling of ligaments including collagen gene expressions. Mechanical stretching signals are mainly transduced by cell adhesion molecules such as integrins. However, the relationships between stress-induced collagen expressions and integrin-mediated cellular behaviors are still unclear in anterior cruciate ligament cells. Here, we focused on the stretch-related responses of different cells derived from the ligament-to-bone interface and midsubstance regions of human anterior cruciate ligaments. Chondroblastic interface cells easily lost their potential to produce collagen genes in non-stretched conditions, rather than fibroblastic midsubstance cells. Uni-axial mechanical stretches increased the type I collagen gene expression of interface and midsubstance cells up to 14- and 6-fold levels of each non-stretched control, respectively. Mechanical stretches also activated the stress fiber formation by shifting the distribution of integrin alphaVbeta3 to the peripheral edges in both interface and midsubstance cells. In addition, integrin alphaVbeta3 colocalized with phosphorylated focal adhesion kinase in stretched cells. Functional blocking analyses using anti-integrin antibodies revealed that the stretch-activated collagen gene expressions on fibronectin were dependent on integrin alphaVbeta3-mediated cellular adhesions in the interface and midsubstance cells. These findings suggest that the integrin alphaVbeta3-mediated stretch signal transduction might have a key role to stimulate collagen gene expression in human anterior cruciate ligament, especially in the ligament-to-bone interface.

Inner meniscus cells maintain higher chondrogenic phenotype compared with outer meniscus cells.

Meniscus cells have several distinct properties in cellular morphology and extracellular matrix production. Inner meniscus cells are considered to have more chondrocytic phenotype compared with outer meniscus cells. However, the chondrogenic property of each meniscus cell has not been elucidated in detail. In this study, we investigated the difference between human inner and outer meniscus-derived cells in extracellular matrix deposition and chondrogenic potential. Monolayer-cultured inner meniscus cells showed small and ovoid shapes though slender and fibroblastic cells were obtained from outer half of human meniscus. The syntheses of type II collagen and safranin O-stained proteoglycans were increased in chondrogenic pellets derived from inner meniscus cells, rather than in outer meniscus cell-derived pellets. On the other hand, adipogenic lipid vacuoles were equally accumulated in both inner and outer meniscus cells after adipogenic treatment. Chondrogenic treatments also enhanced the expression of chondrogenic marker genes, such as Sry-type HMG box (SOX) 9, Scleraxis, and α1(II) collagen, in inner meniscus cells. However, SOX9 expression was not increased in outer meniscus cells even after chondrogenic treatment. This study demonstrated that inner meniscus cells maintained higher chondrogenic potential compared with outer meniscus cells. Our results suggest that the difference between inner and outer meniscus cells in chondrogenic property might have an essential role in preserving a zone-specific meniscal feature.