Small-molecule probe reveals a kinase cascade that links stress signaling to TCF/LEF and Wnt responsiveness.

Wnt signaling plays a central role in tissue maintenance and cancer. Wnt activates downstream genes through ß-catenin, which interacts with TCF/LEF transcription factors. A major question is how this signaling is coordinated relative to tissue organization and renewal. We used a recently described class of small molecules that binds tubulin to reveal a molecular cascade linking stress signaling through ATM, HIPK2, and p53 to the regulation of TCF/LEF transcriptional activity. These data suggest a mechanism by which mitotic and genotoxic stress can indirectly modulate Wnt responsiveness to exert coherent control over cell shape and renewal. These findings have implications for understanding tissue morphogenesis and small-molecule anticancer therapeutics.

Coordinate Nodal and BMP inhibition directs Baf60c-dependent cardiomyocyte commitment.

A critical but molecularly uncharacterized step in heart formation and regeneration is the process that commits progenitor cells to differentiate into cardiomyocytes. Here, we show that the endoderm-derived dual Nodal/bone morphogenetic protein (BMP) antagonist Cerberus-1 (Cer1) in embryonic stem cell cultures orchestrates two signaling pathways that direct the SWI/SNF chromatin remodeling complex to cardiomyogenic loci in multipotent (KDR/Flk1+) progenitors, activating lineage-specific transcription. Transient inhibition of Nodal by Cer1 induces Brahma-associated factor 60c (Baf60c), one of three Baf60 variants (a, b, and c) that are mutually exclusively assembled into SWI/SNF. Blocking Nodal and BMP also induces lineage-specific transcription factors Gata4 and Tbx5, which interact with Baf60c. siRNA to Cer1, Baf60c, or the catalytic SWI/SNF subunit Brg1 prevented the developmental opening of chromatin surrounding the Nkx2.5 early cardiac enhancer and cardiomyocyte differentiation. Overexpression of Baf60c fully rescued these deficits, positioning Baf60c and SWI/SNF function downstream from Cer1. Thus, antagonism of Nodal and BMP coordinates induction of the myogenic Baf60c variant and interacting transcription factors to program the developmental opening of cardiomyocyte-specific loci in chromatin. This is the first demonstration that cues from the progenitor cell environment direct the subunit variant composition of SWI/SNF to remodel the transcriptional landscape for lineage-specific differentiation.

Wnt inhibition correlates with human embryonic stem cell cardiomyogenesis: a structure-activity relationship study based on inhibitors for the Wnt response.

Human embryonic stem cell-based high-content screening of 550 known signal transduction modulators showed that one "lead" (1, a recently described inhibitor of the proteolytic degradation of Axin) stimulated cardiomyogenesis. Because Axin controls canonical Wnt signaling, we conducted an investigation to determine whether the cardiogenic activity of 1 is Wnt-dependent, and we developed a structure-activity relationship to optimize the cardiogenic properties of 1. We prepared analogues with a range of potencies (low nanomolar to inactive) for Wnt/ß-catenin inhibition and for cardiogenic induction. Both functional activities correlated positively (r(2) = 0.72). The optimal compounds induced cardiogenesis 1.5-fold greater than 1 at 30-fold lower concentrations. In contrast, no correlation was observed for cardiogenesis and modulation of transforming growth factor ß (TGFß)/Smad signaling that prominently influences cardiogenesis. Taken together, these data show that Wnt signaling inhibition is essential for cardiogenic activity and that the pathway can be targeted for the design of druglike cardiogenic molecules.

Stage-specific secretion of HMGB1 in cartilage regulates endochondral ossification.

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.

Sox9 and p300 cooperatively regulate chromatin-mediated transcription.

Chromatin structure is a fundamental component of gene regulation, expression, and cellular differentiation. We have previously reported that the multifunctional coactivator p300 is a member of the Sox9 (Sry-type high mobility group box 9)-related transcriptional apparatus and activates Sox9-dependent transcription during chondrogenesis. However, the mechanism of synergy between Sox9 and p300 in chromatin-mediated transcription has not been elucidated. In the present study we investigated the activity of Sox9 and p300 on chromatinized templates in vitro. Recombinant Sox9 was shown to be associated with several transcriptional cofactors including p300. In vitro transcription assays revealed that p300 potentiated Sox9-dependent transcription on chromatinized DNA and, importantly, was associated with hyperacetylated histones. Consistent with these results, the histone deacetylase inhibitor trichostatin A stimulated the expression of Sox9-regulated cartilage matrix genes and induced histone acetylation around the enhancer region of the collagen alpha1 (II) gene in chondrocytes. These findings suggest that Sox9 interacts with chromatin and activates transcription via regulation of chromatin modification.

Transcriptional coactivator PGC-1alpha regulates chondrogenesis via association with Sox9.

Chondrogenesis is a multistep pathway in which multipotential mesenchymal stem cells (MSC) differentiate into chondrocytes. The transcription factor Sox9 (SRY-related high mobility group-Box gene 9) regulates chondrocyte differentiation and cartilage-specific expression of genes, such as Col2a1 (collagen type II alpha1). However, Sox9 expression is detected not only in chondrogenic tissue but also in nonchondrogenic tissues, suggesting the existence of a molecular partner(s) required for Sox9 to control chondrogenesis and chondrogenic gene expression. Here, we report identification of peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1alpha) as a coactivator for Sox9 during chondrogenesis. Expression of PGC-1alpha is induced at chondrogenesis sites during mouse embryonic limb development and during chondrogenesis in human MSC cultures. PGC-1alpha directly interacts with Sox9 and promotes Sox9-dependent transcriptional activity, suggesting that PGC-1alpha acts as a transcriptional coactivator for Sox9. Consistent with this finding, PGC-1alpha disruption in MSC by small interfering RNA inhibits Col2a1 expression during chondrogenesis. Furthermore, overexpression of both PGC-1alpha and Sox9 induced expression of chondrogenic genes, including Col2a1, followed by chondrogenesis in the MSC and developing chick limb. Together, our results suggest a transcriptional mechanism for chondrogenesis that is coordinated by PGC-1alpha.

Smad3 induces chondrogenesis through the activation of SOX9 via CREB-binding protein/p300 recruitment.

The transcriptional activation by SRY-type high mobility group box 9 (SOX9) and the transforming growth factor beta (TGF-beta) signals are necessary for chondrogenic differentiation. We have previously shown that CREB-binding protein (CBP/p300) act as an important SOX9 co-activator during chondrogenesis. In the present study, we investigated the relationship between TGF-beta-dependent Smad2/3 signaling pathways and the SOX9-CBP/p300 transcriptional complex at the early stage of chondrogenesis. Overexpressed Smad3 strongly induced the primary chondrogenesis of human mesenchymal stem cells. In addition, Smad3 enhanced the transcriptional activity of SOX9 and the expression of alpha1(II) collagen gene (COL2A1), and small interference RNA against Smad3 (si-Smad3) inhibited them. We observed that Smad2/3 associated with Sox9 in a TGF-beta-dependent manner and formed the transcriptional complexes with SOX9 on the enhancer region of COL2A1. Interestingly, the association between Sox9 and CBP/p300 was increased by Smad3 overexpression and was suppressed by si-Smad3. Our findings indicate that Smad3 has a stronger potential to stimulate the SOX9-dependent transcriptional activity by modulating the interaction between SOX9 and CBP/p300, rather than Smad2. This study suggests that the Smad3 pathway presents a key role for the SOX9-dependent transcriptional activation in primary chondrogenesis.

HTLV-I env protein acts as a major antigen in patients with HTLV-I-associated arthropathy.

Our objective was to investigate the pathological mechanisms of HTLV-I (human T-cell leukemia virus type I)-associated chronic arthritis (HAAP) with respect to T-cell response to HTLV-I viral proteins. We examined T-cell clonality and the antigen recognized by T cells from the inflamed synovium of patients with HAAP by using histology, a single-strand conformation polymorphism (SSCP) analysis and T cell receptor (TCR) sequencing. The SSCP analysis showed oligoclonal expansion of T cells in the synovium, suggesting an antigen-mediated stimulation. In contrast, there was less clonal expansion in peripheral blood lymphocytes (PBL). The expression of HTLV-1 env and tax mRNA was detected in the affected synovium as well as in PBL. A number of T-cell clones in the synovium recognized HTLV-I env and tax proteins. Twenty-seven (24.9%) of 109 examined T-cell clones in the joints were HTLV-I env reactive, and 7 clones (6.4%) were HTLV-I tax reactive. Junctional sequence analysis of synovial T cells showed a lack of highly conserved amino acid motifs in the complementarity-determining region 3 (CDR3) of HTLV-I env and tax reactive T cells, suggesting that these cells recognized multiple T-cell epitopes on HTLV-I antigen. These findings suggest that HTLV-I env protein acts as a major antigen and may play a role in the development of arthropathy in patients with HAAP.

Transcriptional co-activators CREB-binding protein and p300 regulate chondrocyte-specific gene expression via association with Sox9.

Chondrocytes are critical components for the precise patterning of a developing skeletal framework and articular joint formation. Sox9 is a key transcription factor that is essential for chondrocyte differentiation and chondrocyte-specific gene expressions; however, the precise transcriptional activation mechanism of Sox9 is not fully understood. Here we demonstrate that Sox9 utilizes a cAMP-response element-binding protein (CREB)-binding protein (CBP)/p300 to exert its effects. Sox9 associates with CBP/p300 in the chondrosarcoma cell line SW1353 via its carboxyl termini activation domain in a cell type-specific manner. In promoter assays, CBP/p300 enhances Col2a1, which encodes cartilage-specific type II collagen gene promoter activity via Sox9. Chromatin immunoprecipitation shows that p300 is bound to the Col2a1 promoter region. Furthermore, the CBP/Sox9 complex disrupter peptide suppresses Col2a1 gene expression and chondrogenesis from mesenchymal stem cells. These data demonstrate that CBP and p300 function as co-activators of Sox9 for cartilage tissue-specific gene expression and chondrocyte differentiation.