Postnatal Sox6 Regulates Synaptic Function of Cortical Parvalbumin-Expressing Neurons.

Cortical parvalbumin-expressing (Pvalb+) neurons provide robust inhibition to neighboring pyramidal neurons, crucial for the proper functioning of cortical networks. This class of inhibitory neurons undergoes extensive synaptic formation and maturation during the first weeks after birth and continue to dynamically maintain their synaptic output throughout adulthood. While several transcription factors, such as Nkx2-1, Lhx6, and Sox6, are known to be necessary for the differentiation of progenitors into Pvalb+ neurons, which transcriptional programs underlie the postnatal maturation and maintenance of Pvalb+ neurons' innervation and synaptic function remains largely unknown. Because Sox6 is continuously expressed in Pvalb+ neurons until adulthood, we used conditional knock-out strategies to investigate its putative role in the postnatal maturation and synaptic function of cortical Pvalb+ neurons in mice of both sexes. We found that early postnatal loss of Sox6 in Pvalb+ neurons leads to failure of synaptic bouton growth, whereas later removal in mature Pvalb+ neurons in the adult causes shrinkage of already established synaptic boutons. Paired recordings between Pvalb+ neurons and pyramidal neurons revealed reduced release probability and increased failure rate of Pvalb+ neurons' synaptic output. Furthermore, Pvalb+ neurons lacking Sox6 display reduced expression of full-length tropomyosin-receptor kinase B (TrkB), a key modulator of GABAergic transmission. Once re-expressed in neurons lacking Sox6, TrkB was sufficient to rescue the morphologic synaptic phenotype. Finally, we showed that Sox6 mRNA levels were increased by motor training. Our data thus suggest a constitutive role for Sox6 in the maintenance of synaptic output from Pvalb+ neurons into adulthood.SIGNIFICANCE STATEMENT Cortical parvalbumin-expressing (Pvalb+) inhibitory neurons provide robust inhibition to neighboring pyramidal neurons, crucial for the proper functioning of cortical networks. These inhibitory neurons undergo extensive synaptic formation and maturation during the first weeks after birth and continue to dynamically maintain their synaptic output throughout adulthood. However, it remains largely unknown which transcriptional programs underlie the postnatal maturation and maintenance of Pvalb+ neurons. Here, we show that the transcription factor Sox6 cell-autonomously regulates the synaptic maintenance and output of Pvalb+ neurons until adulthood, leaving unaffected other maturational features of this neuronal population.

M2 macrophage-derived exosomes carry miR-1271-5p to alleviate cardiac injury in acute myocardial infarction through down-regulating SOX6.

BACKGROUND: Emerging evidence has indicated that exosomes serve as key regulators in acute myocardial infarction (AMI). This study was determined to investigate the effect of M2 macrophage-derived exosomes (M2-Exos) in AMI and the further mechanism. METHODS: M2 macrophages were induced and M2-exos were isolated and verified. The AMI mouse model was prepared by ligation of the left anterior descending coronary artery (LAD) and then intravenously injected with the isolated M2-exos. The mouse cardiac function was assessed by echocardiography. Hematoxylin and eosin (HE) staining and TUNEL assay were conducted to examine myocardial lesion and apoptosis in cardiac tissues. The expressions of associated molecules were detected by quantitative real time-PCR (qRT-PCR) and western blot. MTT assay, Flow cytometry and Dual-luciferase reporter assay were carried out to detect cell viability, apoptosis and the interaction of miRNA and the target. RESULT: M2-Exos could promote cardiac repair in AMI mice. M2-Exos suppressed apoptosis and enhanced viability of hypoxia-induced cardiomyocytes through delivery of miR-1271-5p. SOX6 is a direct target of miR-1271-5p. miR-1271-5p decreased cardiomyocyte apoptosis induced by hypoxia and alleviated cardiac injury in AMI via down-regulating SOX6 expression. CONCLUSION: We identified that M2-Exos could carry miR-1271-5p to reduce apoptosis of cardiomyocytes and promote cardiac repair via down-regulating SOX6.

Blockage of miR-485-5p on Cortical Neuronal Apoptosis Induced by Oxygen and Glucose Deprivation/Reoxygenation Through Inactivating MAPK Pathway.

This study is designed to explore the role of miR-485-5p in hypoxia/reoxygenation-induced neuronal injury in primary rat cortical neurons. Hypoxia/reoxygenation model was established through oxygen and glucose deprivation/reoxygenation (OGD/R). RN-c cells were transfected with miR-485-5p mimics, miR-485-5p inhibitors, si-SOX6, pCNDA3.1-SOX6 or miR-485-5p + pCDNA3.1-SOX6, in which cell viability, apoptosis, lactate dehydrogenase (LDH) release rate were assessed. Western blot detected the protein expressions of apoptotic-related proteins (caspase3, Bcl-2, Bax) and the phosphorylated level of ERK1/2. The potential binding sites between miR-485-5p and SOX6 were predicted by STARBASE and identified using dual luciferase reporter gene assay. OGD/R-treated RN-c cell presented increases in apoptosis and LDH release rate as well as a decrease in cell viability. miR-485-5p was downregulated while SOX6 was upregulated in OGD/R-treated RN-c cells. Overexpression of miR-485-5p or SOX6 knockdown rescued cell viability and Bcl-2 expression, while attenuated apoptosis, LDH release rate, expression of SOX6 and the phosphorylated level of ERK1/2. Consistently, miR-485-5p inhibition led to the reverse pattern. Co-transfection of miR-485-5p and SOX6 reversed the protective effect of miR-485-5p on OGD/R-induced neuronal apoptosis. miR-485-5p can directly target SOX6. Together, miR-485-5p inhibited SOX6 to alleviate OGD/R-induced apoptosis. Collectively, miR-485-5p protects primary cortical neurons against hypoxia injury through downregulating SOX6 and inhibiting MAPK pathway.

High expression levels and localization of Sox5 in dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a disease that can lead to heart expansion and severe heart failure, but the specific pathogenesis remains unclear. Sox5 is a member of the Sox family with a key role in cardiac function. However, the role of Sox5 in DCM remains unclear. In the present study, wild­type mice were intraperitoneally injected with doxorubicin (Dox) to induce DCM, and heart specimens from human patients with DCM were used to investigate the preliminary role of Sox5 in DCM. The present study demonstrated that, compared with control human hearts, the hearts of patients with DCM exhibited high expression levels of Sox5 and activation of the wnt/ß­catenin pathway. This result was consistent with Dox­induced DCM in mice. Furthermore, in Dox­treated mice, apoptosis was activated during the development of DCM. Inflammation and collagen deposition also increased in DCM mice. The results of the present study indicate that Sox5 may be associated with the development of DCM. Sox5 may be a novel potential factor that regulates DCM.

Identification of HMG-box family establishes the significance of SOX6 in the malignant progression of glioblastoma.

Glioblastoma multiforme (GBM) is the most malignant neuroepithelial primary brain tumor and its mean survival time is 15 months after diagnosis. This study undertook to investigate the genome-wide and transcriptome-wide analyses of human high mobility group box (HMG-box) TF (transcript factor) families / HOX, TOX, FOX, HMG and SOX gene families, and their relationships to GBM. According to the TCGA-GBM profile analysis, differentially expressed HOX, FOX, HMG and SOX gene families (62 DEmRNA) were found in this study. We also analyzed DEmRNA (HMG-box related genes) co-expressed eight DElncRNA in GBM, and constructed a ceRNA network analysis as well. We constructed 50 DElncRNA-DEmiRNA-DEmRNA (HMG-box related genes) pairs between GBM and normal tissues. Then, risk genes SOX6 and SOX21 expression were correlated with immune infiltration levels in GBM. SOX6 also had a strong association with MAPT, GSK3B, FYN and DPYSL4, suggesting that they might be functional members in GBM.

Dual functional nanoparticles containing SOX duo and ANGPT4 shRNA for osteoarthritis treatment.

In our previous studies, we found that adult stem cells transfected with sex-determining region Y-box (SOX)-9, -6 and -5 genes (SOX trio) enhanced chondrogenesis and suppressed the progression of osteoarthritis (OA). The inhibition of angiopoietin-like 4 (ANGPT4) is known to reduce levels of cartilage damaging enzymes, such as, matrix metalloproteinases (MMPs). In this study, we designed nanoparticles comprising dexamethasone-conjugated polyethylenimine (DEX PEI) complexed with minicircle plasmid (MC) harboring SOX duo (SOX-9, -6) and ANGPTL4 small hairpin RNA (shANG) [MC SOX9/6/shANG] in the expectation that transfection of these nanoparticles would enhance chondrogenesis of stem cells and suppress inflammation in OA. Adipose-derived stem cells (ADSCs) transfected with MC SOX9/6/shANG (MC SOX9/6/shANG-tADSCs) showed significantly higher expressions of COL2 gene and protein than MC SOX9/6-transfected ADSCs (MC SOX9/6-tADSCs) during in vitro chondrogenesis while both enhanced chondrogenesis in the absence of growth factor addition as compared with negative controls. Furthermore, the expressions of MMP13 and MMP3 genes were significantly more diminished in MC SOX9/6/shANG-tADSCs than in MC SOX9/6-tADSCs. In vivo experiments using surgically-induced OA rats showed MC SOX9/6/shANG-tADSC-treated rats had significantly lower levels of cyclooxygenase (COX-2) and MMP13 in synovial fluids than MC SOX9/6-tADSC-treated rats, but no significant difference was observed between them in histological appearances. Both groups showed significantly less joint destruction than control groups did. These results demonstrate that dual functional nanoparticles containing SOX duo and ANGPT4 shRNA enhance chondrogenesis of ADSCs and suppress inflammation in OA. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:234-242, 2020.

microRNA-181b suppresses the metastasis of lung cancer cells by targeting sex determining region Y-related high mobility group-box 6 (Sox6).

BACKGROUND: The aim of the study was to measure the expression of microRNA (miR)-181b in patients with lung cancer, investigate its biological function and elucidate the underlying mechanisms associated with the development of lung cancer. METHODS: miR-181b expression in tissues was measured via RT-qPCR. After A549 cells were transfected with miR-181b mimic or si-Sox6, the proliferation, migration and cell cycle distribution of A549 were evaluated using cell counting kit-8 assay, transwell assay and flow cytometry. The levels of cell cycle-related proteins and Sox6 were analyzed by western blotting. Gene targets of miR-181b were predicted via bioinformatics analysis and verified using a dual-luciferase reporter gene assay. RESULTS: Expression of miR-181b was significantly downregulated in lung cancer tissues (P < 0.05), and was inversely correlated with the degree of cell differentiation and clinical stages of lung cancer (both P < 0.05). Additionally, the expression of miR-181b was significantly lower in adenocarcinoma compared with squamous cell carcinoma in the lungs (P < 0.05). Overexpression of miR-181b significantly decreased the protein level of Sox6 and significantly suppressed the cell proliferation and metastasis (both P < 0.05); this effect was also observed in A549 cells transfected with si-Sox6. The luciferase activity of a Sox6 3'-untranslated region-based reporter construct was significantly lower when transfected with miR-181b (P < 0.05), which suggests that Sox6 is a direct target of miR-181b. CONCLUSION: The results of the present study suggest that miR-181b may function as a tumor inhibitor in the development of lung cancer via targeting Sox6 to decrease the proliferation and metastasis of lung cancer cells.

MiR-1269a acts as an onco-miRNA in non-small cell lung cancer via down-regulating SOX6.

OBJECTIVE: Lung cancer, especially non-small cell lung cancer (NSCLC), remains one of the leading death-causing malignant tumors worldwide. MicroRNAs (miRNAs) have been identified to participate in the development and progression of NSCLC. However, the role of miR-1269a in NSCLC still needs to be elucidated. The objective of this study was to investigate the function of miR-1269a in NSCLC and its underlying mechanism. PATIENTS AND METHODS: Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was utilized to measure the expression level of miR-1269a in NSCLC tissues and cell lines. After transfection with miR-1269a mimics or inhibitors, the expression level of miR-1269a in NSCLC was up- or down-regulated. Cell Counting Kit-8 (CCK-8) assay and colony formation assay were used to measure cell proliferation ability. Flow cytometry assay was applied to verify the cell cycle distributions of established cell lines. The potential target of miR-1269a was determined by using dual-luciferase and Western blot assays. RESULTS: miR-1269a was significantly over-expressed in NSCLC tissues than that in adjacent normal tissues. The expression of miR-1269a was also up-regulated in NSCLC-derived cell lines. Up-regulation of miR-1269a improved the abilities of cell proliferation, colony formation, and induced cell cycle transition. Meanwhile, down-regulation of miR-1269a decreased the capacities of cell proliferation, colony formation, and arrested the cell cycle. It was further implicated that SOX6 was verified as a target of miR-1269a in NSCLC and over-expressed SOX6 could rescue the effect of miR-1269a up-regulation. CONCLUSIONS: Our study demonstrated that miR-1299a could function as an onco-miRNA in NSCLC and promote NSCLC growth via down-regulating the expression of SOX6.

TNF-α-Induced SOX5 Upregulation Is Involved in the Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells Through KLF4 Signal Pathway.

Postmenopausal osteoporosis (PMOP) is a common systemic skeletal disease characterized by reduced bone mass and microarchitecture deterioration. Although differentially expressed SOX5 has been found in bone marrow from ovariectomized mice, its role in osteogenic differentiation in human mesenchymal stem cells (hMSCs) from bone marrow in PMOP remains unknown. In this study, we investigated the biological function of SOX5 and explore its molecular mechanism in hMSCs from patients with PMOP. Our findings showed that the mRNA and protein expression levels of SOX5 were upregulated in hMSCs isolated from bone marrow samples of PMOP patients. We also found that SOX5 overexpression decreased the alkaline phosphatase (ALP) activity and the gene expression of osteoblast markers including Collagen I, Runx2 and Osterix, which were increased by SOX5 knockdown using RNA interference. Furthermore, TNF-α notably upregulated the SOX5 mRNA expression level, and SOX5 knockdown reversed the effect of TNF-α on osteogenic differentiation of hMSCs. In addition, SOX5 overexpression increased Kruppel-like factor 4 (KLF4) gene expression, which was decreased by SOX5 silencing. KLF4 knockdown abrogated the suppressive effect of SOX5 overexpression on osteogenic differentiation of hMSCs. Taken together, our results indicated that TNF-α-induced SOX5 upregulation inhibited osteogenic differentiation of hMSCs through KLF4 signal pathway, suggesting that SOX5 might be a novel therapeutic target for PMOP treatment.

Sox5 is involved in germ-cell regulation and sex determination in medaka following co-option of nested transposable elements.

BACKGROUND: Sex determination relies on a hierarchically structured network of genes, and is one of the most plastic processes in evolution. The evolution of sex-determining genes within a network, by neo- or sub-functionalization, also requires the regulatory landscape to be rewired to accommodate these novel gene functions. We previously showed that in medaka fish, the regulatory landscape of the master male-determining gene dmrt1bY underwent a profound rearrangement, concomitantly with acquiring a dominant position within the sex-determining network. This rewiring was brought about by the exaptation of a transposable element (TE) called Izanagi, which is co-opted to act as a silencer to turn off the dmrt1bY gene after it performed its function in sex determination. RESULTS: We now show that a second TE, Rex1, has been incorporated into Izanagi. The insertion of Rex1 brought in a preformed regulatory element for the transcription factor Sox5, which here functions in establishing the temporal and cell-type-specific expression pattern of dmrt1bY. Mutant analysis demonstrates the importance of Sox5 in the gonadal development of medaka, and possibly in mice, in a dmrt1bY-independent manner. Moreover, Sox5 medaka mutants have complete female-to-male sex reversal. CONCLUSIONS: Our work reveals an unexpected complexity in TE-mediated transcriptional rewiring, with the exaptation of a second TE into a network already rewired by a TE. We also show a dual role for Sox5 during sex determination: first, as an evolutionarily conserved regulator of germ-cell number in medaka, and second, by de novo regulation of dmrt1 transcriptional activity during primary sex determination due to exaptation of the Rex1 transposable element.

The role of Sox6 and Netrin-1 in ovarian cancer cell growth, invasiveness, and angiogenesis.

SOX6 plays important roles in cell proliferation, differentiation, and cell fate determination. It has been confirmed that SOX6 is a tumor suppressor and downregulated in various cancers, including esophageal squamous cell carcinoma, hepatocellular carcinoma, and chronic myeloid leukemia. Netrin-1 is highly expressed in various human cancers and acts as an anti-apoptotic and proangiogenic factor to drive tumorigenesis. The role of SOX6 and netrin-1 in regulating the growth of ovarian tumor cells still remains unclear. Real-time polymerase chain reaction and western blot were used to determine the SOX6 messenger RNA and protein levels, respectively, in ovarian cancer cells and tumor tissues. Stable transfection of SOX6 was conducted to overexpress SOX6 in PA-1 and SW626 cells. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Invasion of ovarian cancer cells and migration of human umbilical vein endothelial cells were confirmed by Transwell assays. To overexpress netrin-1, ovarian cancer cells with SOX6 restoration was transduced with netrin-1 lentiviral particles. PA-1 xenografts in a nude mice model were used to conduct in vivo evaluation of the role of SOX6 and its relationship with netrin-1 in tumor growth and angiogenesis. In this study, we found significantly reduced SOX6 levels in PA-1, SW626, SK-OV-3, and CaoV-3 ovarian cancer cell lines and human tumor tissues in comparison with normal human ovarian epithelial cells or matched non-tumor tissues. SOX6 overexpression by stable transfection dramatically inhibited proliferation and invasion of PA-1 and SW626 cells. Also, conditioned medium from PA-1 and SW626 cells with SOX6 restoration exhibited reduced ability to induce human umbilical vein endothelial cells migration and tube formation compared with conditioned medium from the cells with transfection control. Furthermore, an inverse relationship between SOX6 and netrin-1 expression was observed in PA-1 and SW626 cells. Overexpression of netrin-1 in ovarian cancer cells with forced SOX6 expression remarkably abrogated the inhibitory effect of SOX6 on proliferation, invasion of the cells, and tumor xenograft growth and vascularity in vivo. Human umbilical vein endothelial cell migration and tube formation were enhanced in the conditioned medium from the ovarian cancer cells transduced with netrin-1 lentivirus particles. Our observations revealed that SOX6 is a tumor suppressor in ovarian cancer cells, and SOX6 exerts an inhibitory effect on the proliferation, invasion, and tumor cell-induced angiogenesis of ovarian cancer cells, whereas nerin-1 plays an opposite role and its expression is inversely correlated with SOX6. Moreover, our findings suggest a new role of SOX6 and netrin-1 for understanding the progression of ovarian cancer and have the potential for the development of new diagnosis and treatment strategies for ovarian cancer.

Role of microRNA-21 in the formation of insulin-producing cells from pancreatic progenitor cells.

MicroRNAs (miRNAs) regulate insulin secretion, pancreas development, and beta cell differentiation. In this study, to screen for miRNAs and their targets that function during insulin-producing cells (IPCs) formation, we examined the messenger RNA and microRNA expression profiles of pancreatic progenitor cells (PPCs) and IPCs using microarray and deep sequencing approaches, respectively. Combining our data with that from previous reports, we found that miR-21 and its targets play an important role in the formation of IPCs. However, the function of miR-21 in the formation of IPCs from PPCs is poorly understood. Therefore, we over-expressed or inhibited miR-21 and expressed small interfering RNAs of miR-21 targets in PPCs to investigate their functions in IPCs formation. We found that miR-21 acts as a bidirectional switch in the formation of IPCs by regulating the expression of target and downstream genes (SOX6, RPBJ and HES1). Small interfering RNAs were used to knock down these genes in PPCs to investigate their effects on IPCs formation. Single expression of si-RBPJ, si-SOX6 and si-HES1 in PPCs showed that si-RBPJ was an inhibitor, and that si-SOX6 and si-HES1 were promoters of IPCs formation, although si-HES1 induced formation of IPCs at higher rates than si-SOX6. These results suggest that endogenous miRNAs involved in the formation of IPCs from PPCs should be considered in the development of an effective cell transplant therapy for diabetes.

Identification of the SOX5 gene as a novel IGH-involved translocation partner in BCL2-negative follicular lymphoma with t(12;14)(p12.2;q32).

Chromosome translocations involving the immunoglobulin heavy chain (IGH) gene locus at chromosome region 14q32 are often observed in B-cell lymphoid neoplasms. Of these, t(14;18)(q32;q21) results in juxtaposition of the IGH gene on chromosome 14 and the BCL2 gene on chromosome 18, leading to the overexpression of BCL2 anti-apoptotic protein, which plays a critical role in the development of follicular lymphoma (FL). However, BCL2 overexpression is not observed in approximately 10 % of FL, and the molecular pathogenesis of BCL2-negative FL has not been elucidated. Here, we identify the SRY-related high-morbidity-group (HMG) box 5 (SOX5) gene on chromosome 12p12 as a novel IGH-involved translocation partner in the case of BCL2-negative follicular lymphoma (FL) with a complex karyotype including t(12;14)(p12.2;q32) by long-distance inverse PCR. As a result of this translocation, the SOX5 gene is juxtaposed to the enhancer of the IGH gene; SOX5 overexpression in neoplastic cells was demonstrated by immunohistochemistry. The results of the present study suggest a role for SOX5 in the molecular pathogenesis of FL.

Identification of a transient Sox5 expressing progenitor population in the neonatal ventral forebrain by a novel cis-regulatory element.

Precise control of lineage-specific gene expression in the neural stem/progenitor cells is crucial for generation of the diversity of neuronal and glial cell types in the central nervous system (CNS). The mechanism underlying such gene regulation, however, is not fully elucidated. Here, we report that a 377 bp evolutionarily conserved DNA fragment (CR5), located approximately 32 kbp upstream of Olig2 transcription start site, acts as a cis-regulator for gene expression in the development of the neonatal forebrain. CR5 is active in a time-specific and brain region-restricted manner. CR5 activity is not detected in the embryonic stage, but it is exclusively in a subset of Sox5+ cells in the neonatal ventral forebrain. Furthermore, we show that Sox5 binding motif in CR5 is important for this cell-specific gene regulatory activity; mutation of Sox5 binding motif in CR5 alters reporter gene expression with different cellular composition. Together, our study provides new insights into the regulation of cell-specific gene expression during CNS development.

Decreased expression of SOX6 confers a poor prognosis in hepatocellular carcinoma.

BACKGROUND AND AIM: SOX6, a member of the D subfamily of sex determining region y-related transcription factors, plays critical roles in cell fate determination, differentiation and proliferation. It has been identified as a tumor suppressor or an oncogene in different human cancers. However, the role of SOX6 in the development and progression of hepatocellular carcinoma (HCC) has not been explored. The aim of this study was to investigate the expression of SOX6 in HCC and determine its correlation with tumor progression and prognosis. METHODS: 130 HCC patients who had undergone curative liver resection were selected and immunohistochemistry, Western blotting, and quantitative real time polymerase chain reaction (Q-PCR) were performed to analyze SOX6 expression in the respective tumors. RESULTS: Q-PCR, immunohistochemistry and Western blotting consistently confirmed the decreased expression of SOX6 at both mRNA and protein levels in HCC tissues compared with their adjacent nonneoplastic tissues (P<0.01). Additionally, the expression of SOX6, determined by immunohistochemistry, was negatively correlated with the tumor stage (P=0.003) and serum AFP (P=0.02). Moreover, HCC patients with lower SOX6 expression had worse 5-year disease-free survival and 5-year overall survival than those with high SOX6 expression (P=0.006 and 0.001, respectively). Furthermore, the Cox proportional hazards model showed that the decreased expression of SOX6 was an independent poor prognostic factor for both 5-year disease-free survival (hazards ratio [HR]=2.398, 95% confidence interval [CI]=1.601-5.993, P=0.01) and 5-year overall survival (HR=3.569, CI=1.381-7.290, P=0.008) in HCC. CONCLUSION: These findings provide evidence for the first time that SOX6 expression was decreased in HCC, which correlated with poor prognosis, suggesting that SOX6 may be a novel and potential prognostic marker for HCC.

Characterization of the DNA-binding properties of the Mohawk homeobox transcription factor.

The homeobox transcription factor Mohawk (Mkx) is a potent transcriptional repressor expressed in the embryonic precursors of skeletal muscle, cartilage, and bone. MKX has recently been shown to be a critical regulator of musculoskeletal tissue differentiation and gene expression; however, the genetic pathways through which MKX functions and its DNA-binding properties are currently unknown. Using a modified bacterial one-hybrid site selection assay, we determined the core DNA-recognition motif of the mouse monomeric Mkx homeodomain to be A-C-A. Using cell-based assays, we have identified a minimal Mkx-responsive element (MRE) located within the Mkx promoter, which is composed of a highly conserved inverted repeat of the core Mkx recognition motif. Using the minimal MRE sequence, we have further identified conserved MREs within the locus of Sox6, a transcription factor that represses slow fiber gene expression during skeletal muscle differentiation. Real-time PCR and immunostaining of in vitro differentiated muscle satellite cells isolated from Mkx-null mice revealed an increase in the expression of Sox6 and down-regulation of slow fiber structural genes. Together, these data identify the unique DNA-recognition properties of MKX and reveal a novel role for Mkx in promoting slow fiber type specification during skeletal muscle differentiation.

The protein kinase MLTK regulates chondrogenesis by inducing the transcription factor Sox6.

Sox9 acts together with Sox5 or Sox6 as a master regulator for chondrogenesis; however, the inter-relationship among these transcription factors remains unclear. Here, we show that the protein kinase MLTK plays an essential role in the onset of chondrogenesis through triggering the induction of Sox6 expression by Sox9. We find that knockdown of MLTK in Xenopus embryos results in drastic loss of craniofacial cartilages without defects in neural crest development. We also find that Sox6 is specifically induced during the onset of chondrogenesis, and that the Sox6 induction is inhibited by MLTK knockdown. Remarkably, Sox6 knockdown phenocopies MLTK knockdown. Moreover, we find that ectopic expression of MLTK induces Sox6 expression in a Sox9-dependent manner. Our data suggest that p38 and JNK pathways function downstream of MLTK during chondrogenesis. These results identify MLTK as a novel key regulator of chondrogenesis, and reveal its action mechanism in chondrocyte differentiation during embryonic development.

A new chondrogenic differentiation initiator with the ability to up-regulate SOX trio expression.

During random screening for chondrogenic differentiation inducers, we found that Compound-1, 4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-1H-pyrazol-3-yl]benzene-1,3-diol, initiated chondrogenic differentiation of the chondroprogenitor cell line ATDC5. Compound-1 initiated chondrogenic differentiation of the mesenchymal stem cell line C3H10T1/2 in regions where cell aggregates formed and simultaneously inhibited adipogenic differentiation. In C3H10T1/2 cells, Compound-1 increased the expression of Sry-related high-mobility-group box transcription factors L-SOX5, SOX6, and SOX9 (SOX trio) more strongly than bone morphogenic protein (BMP)-2. cAMP-dependent protein kinase (PKA) inhibitors suppressed Compound-1-dependent L-SOX5 and SOX6 up-regulation. PKA inhibitors also suppressed the up-regulation of aggrecan mRNA induced by Compound-1, indicating that increases in L-SOX5 and SOX6 mRNA, in which the PKA pathway participates, are involved in the mechanisms behind the action of Compound-1. On the other hand, the SOX6 and aggrecan gene expression, which were up-regulated by BMP-2, were not affected by the PKA inhibitor. Compound-1 induced chondrogenic differentiation of bone marrow stromal cells and recovered cartilage matrix production by primary chondrocytes, which had been decreased by interleukin-1beta. These results show the potential of Compound-1 to be a new cartilage repair agent for inducing chondrogenic differentiation via SOX trio up-regulation.