RESUMEN

Broadening of signals from atoms at interfaces can often be a limiting factor in applying solution NMR to the structure determination of complexes. Common contributors to such problems include exchange between free and bound states and the increased molecular weight of complexes relative to the free components, but another cause that can be more difficult to deal with occurs when conformational dynamics within the interface takes place at an intermediate rate on the chemical shift timescale. In this work we show how a carefully chosen mutation in the protein HMG-D rescued such a situation, making possible high-resolution structure determination of its complex with a dA2 bulge DNA ligand designed to mimic a natural DNA bend, and thereby revealing a new spatial organization of the complex.

RESUMEN

The dimorphic yeast Yarrowia lipolytica possesses an excellent ability to utilize n-alkane as a sole carbon and energy source. Although there are detailed studies on the enzymes that catalyze the reactions in the metabolic processes of n-alkane in Y. lipolytica, the molecular mechanism underlying the incorporation of n-alkane into the cells remains to be elucidated. Because Y. lipolytica adsorbs n-alkane, we postulated that Y. lipolytica incorporates n-alkane through direct interaction with it. We isolated and characterized mutants defective in adsorption to n-hexadecane. One of the mutants harbored a nonsense mutation in MAR1 (Morphology and n-alkane Adsorption Regulator 1) encoding a protein containing a high mobility group box. The deletion mutant of MAR1 exhibited defects in adsorption to n-hexadecane and filamentous growth on solid media, whereas the strain that overexpressed MAR1 exhibited hyperfilamentous growth. Fluorescence microscopic observations suggested that Mar1 localizes in the nucleus. RNA-sequencing analysis revealed the alteration of the transcript levels of several genes, including those encoding transcription factors and cell surface proteins, by the deletion of MAR1. These findings suggest that MAR1 is involved in the transcriptional regulation of the genes required for n-alkane adsorption and cell morphology transition.IMPORTANCEYarrowia lipolytica, a dimorphic yeast capable of assimilating n-alkane as a carbon and energy source, has been extensively studied as a promising host for bioconversion of n-alkane into useful chemicals and bioremediation of soil and water contaminated by petroleum. While the metabolic pathway of n-alkane in this yeast and the enzymes involved in this pathway have been well characterized, the molecular mechanism to incorporate n-alkane into the cells is yet to be fully understood. Due to the ability of Y. lipolytica to adsorb n-alkane, it has been hypothesized that Y. lipolytica incorporates n-alkane through direct interaction with it. In this study, we identified a gene, MAR1, which plays a crucial role in the transcriptional regulation of the genes necessary for the adsorption to n-alkane and the transition of the cell morphology in Y. lipolytica. Our findings provide valuable insights that could lead to advanced applications of Y. lipolytica in n-alkane bioconversion and bioremediation.

Asunto(s)

RESUMEN

SOX proteins are a family of transcription factors (TFs) that play critical functions in sex determination, neurogenesis, and chondrocyte differentiation, as well as cardiac, vascular, and lymphatic development. There are 20 SOX family members in humans, each sharing a 79-residue L-shaped high mobility group (HMG)-box domain that is responsible for DNA binding. SOX2 was recently shown to interact with long non-coding RNA and large-intergenic non-coding RNA to regulate embryonic stem cell and neuronal differentiation. The RNA binding region was shown to reside within the HMG-box domain; however, the structural details of this binding remain unclear. Here, we show that all SOX family members, except group H, interact with RNA. Our mutational experiments demonstrate that the disordered C-terminal region of the HMG-box domain plays an important role in RNA binding. Further, by determining a high-resolution structure of the HMG-box domain of the group H family member SOX30, we show that despite differences in RNA binding ability, SOX30 shares a very similar secondary structure with other SOX protein HMG-box domains. Together, our study provides insight into the interaction of SOX TFs with RNA.

Asunto(s)

RESUMEN

Cardiac hypertrophy is an adaptive response to stressors such as high cardiac workload, which might lead to abnormal cardiac function and heart failure. Previous studies have indicated that macrophage migration inhibitory factor (MIF) might play a protective role in cardiac hypertrophy. Here, we aimed to illustrate the mechanism of MIF in protecting against pressure overload-induced cardiac hypertrophy. Transverse aortic constriction (TAC) mouse model was established and we found that overexpression of MIF protected against pressure overload-induced cardiac hypotrophy in TAC treated mice, as evidenced by significantly decreased the heart weight. In addition, transthoracic echocardiography showed that overexpression of MIF restored ejection fraction in TAC-treated mice. While TAC treatment resulted in a much larger cardiomyocyte size in mice, MIF overexpression notably decreased the cardiomyocyte size. Next, we demonstrated that MIF overexpression promoted the expression of miR-29b-3p which further downregulated the expression of its downstream target HMG box protein 1 (HBP1). Overexpression of HBP1 reversed the effect of MIF in alleviating Ang-II induced oxidative stress in cardiomyocytes. In conclusion, our findings suggest that MIF could attenuate pressure overload-induced cardiac hypertrophy through regulating the miR-29b-3p/HBP1 axis.

Asunto(s)

RESUMEN

Background: SRY-related HMG-box 10 (SOX10) protein has a confirmed role in the regulation of neural cell proliferation and differentiation. It is now suggested that the changes in SOX10 expression may be linked to neural invasion by cancer cells. We aimed to assess the value of SOX10 expression in predicting perineural invasion in gastric cancer. Methods: A cross-sectional study was performed on 40 patients with gastric cancer. To assess perineural invasion, Hematoxylin & Eosin stained slides were examined. The expression of SOX10 was also examined by immunohistochemistry. Results: Our study showed higher perineural invasion in those with SOX10 positivity as compared to those without SOX10 expression (64.0% vs. 6.7%, p=0.001). No association was revealed between other baseline variables and SOX10 positivity. The expression of this marker increased the chance of neural invasion up to 17 times as indicated by the multivariable regression modeling. Multivariable regression modeling indicated that the chance of neural invasion increased up to 17 times in cases of SOX10 positivity. Conclusion: Overexpression of SOX10 is closely associated with the risk of perineural invasion in gastric cancer.(AU)

Antecedentes: La proteína HMG-box 10 (SOX10) relacionada con SRY tiene un papel confirmado en la regulación de la proliferación y diferenciación de células neurales. Ahora se propone que los cambios en la expresión de SOX10 pueden estar relacionados con la invasión neuronal de las células cancerosas. Nuestro objetivo fue evaluar el valor de la expresión de SOX10 en la predicción de la invasión perineural en el cáncer gástrico. Métodos: Este estudio transversal se realizó en 40 pacientes con cánceres gástricos. Para evaluar la invasión perineural, se planificó la evaluación histopatológica mediante tinción con hematoxilina y eosina. El estado de expresión de SOX10 también se examinó mediante la técnica de inmunohistoquímica. Resultados: Nuestro estudio mostró una mayor invasión perineural en aquellos con positividad de SOX10 en comparación con aquellos sin expresión de SOX10 (64,0% versus 6,7%; p=0,001). No se reveló ninguna asociación entre otras variables basales y la positividad de SOX10. La expresión de este marcador aumentó la posibilidad de invasión neural hasta 17 veces, como indica el modelo de regresión multivariable. Conclusión: La sobreexpresión de SOX10 está estrechamente asociada con el riesgo de invasión perineural en el cáncer gástrico.(AU)

Asunto(s)

RESUMEN

BACKGROUND: SRY-related HMG-box 10 (SOX10) protein has a confirmed role in the regulation of neural cell proliferation and differentiation. It is now suggested that the changes in SOX10 expression may be linked to neural invasion by cancer cells. We aimed to assess the value of SOX10 expression in predicting perineural invasion in gastric cancer. METHODS: A cross-sectional study was performed on 40 patients with gastric cancer. To assess perineural invasion, Hematoxylin & Eosin stained slides were examined. The expression of SOX10 was also examined by immunohistochemistry. RESULTS: Our study showed higher perineural invasion in those with SOX10 positivity as compared to those without SOX10 expression (64.0% vs. 6.7%, p=0.001). No association was revealed between other baseline variables and SOX10 positivity. The expression of this marker increased the chance of neural invasion up to 17 times as indicated by the multivariable regression modeling. Multivariable regression modeling indicated that the chance of neural invasion increased up to 17 times in cases of SOX10 positivity. CONCLUSION: Overexpression of SOX10 is closely associated with the risk of perineural invasion in gastric cancer.

Asunto(s)

RESUMEN

WD repeat and HMG-box DNA binding protein 1 (WDHD1) is a highly conserved gene from yeast to humans. It actively participates in DNA replication, playing a crucial role in DNA damage repair and the cell cycle, contributing to centromere formation and sister chromosome segregation. Notably, several studies have implicated WDHD1 in the development and progression of diverse tumor types, including esophageal carcinoma, pulmonary carcinoma, and breast carcinoma. Additionally, the inhibitor of WDHD1 has been found to enhance radiation sensitivity, improve drug resistance, and significantly decrease tumor cell proliferation. This comprehensive review aims to provide an overview of the molecular structure, biological functions, and regulatory mechanisms of WDHD1 in tumors, thereby establishing a foundation for future investigations and potential clinical applications of WDHD1.

Asunto(s)

RESUMEN

Vascular smooth muscle cells (VSMCs) can transdifferentiate into macrophage-like cells in the context of sustained inflammatory injury, which drives vascular hyperplasia and atherosclerotic complications. Using single-cell RNA sequencing, we identify that macrophage-like VSMCs are the key cell population in mouse neointimal hyperplasia. Sex-determining region Y (SRY)-related HMG-box gene 10 (Sox10) upregulation is associated with macrophage-like VSMC accumulation and pyroptosis in vitro and in the neointimal hyperplasia of mice. Tumor necrosis factor α (TNF-α)-induced Sox10 lactylation in a phosphorylation-dependent manner by PI3K/AKT signaling drives transcriptional programs of VSMC transdifferentiation, contributing to pyroptosis. The regulator of G protein signaling 5 (RGS5) interacts with AKT and blocks PI3K/AKT signaling and Sox10 phosphorylation at S24. Sox10 silencing mitigates vascular inflammation and forestalls neointimal hyperplasia in RGS5 knockout mice. Collectively, this study shows that Sox10 is a regulator of vascular inflammation and a potential control point in inflammation-related vascular disease.

Asunto(s)

RESUMEN

Considerable evidence now indicates that cognitive impairment is primarily a vascular disorder. The depletion of smooth muscle 22 alpha (SM22α) contributes to vascular smooth muscle cells (VSMCs) switching from contractile to synthetic and proinflammatory phenotypes in the context of inflammation. However, the role of VSMCs in the pathogenesis of cognitive impairment remains undetermined. Herein, we showed a possible link between VSMC phenotypic switching and neurodegenerative diseases via the integration of multi-omics data. SM22α knockout (Sm22α-/-) mice exhibited obvious cognitive impairment and cerebral pathological changes, which were visibly ameliorated by the administration of AAV-SM22α. Finally, we confirmed that SM22α disruption promotes the expression of SRY-related HMG-box gene 10 (Sox10) in VSMCs, thereby aggravating the systemic vascular inflammatory response and ultimately leading to cognitive impairment in the brain. Therefore, this study supports the idea of VSMCs and SM22α as promising therapeutic targets in cognitive impairment to improve memory and cognitive decline.

Asunto(s)

RESUMEN

BACKGROUND: Human accelerated regions (HARs) are short conserved genomic sequences that have acquired significantly more nucleotide substitutions than expected in the human lineage after divergence from chimpanzees. The fast evolution of HARs may reflect their roles in the origin of human-specific traits. A recent study has reported positively-selected single nucleotide variants (SNVs) within brain-exclusive human accelerated enhancers (BE-HAEs) hs1210 (forebrain), hs563 (hindbrain) and hs304 (midbrain/forebrain). By including data from archaic hominins, these SNVs were shown to be Homo sapiens-specific, residing within transcriptional factors binding sites (TFBSs) for SOX2 (hs1210), RUNX1/3 (hs563), and FOS/JUND (hs304). Although these findings suggest that the predicted modifications in TFBSs may have some role in present-day brain structure, work is required to verify the extent to which these changes translate into functional variation. RESULTS: To start to fill this gap, we investigate the SOX2 SNV, with both forebrain expression and strong signal of positive selection in humans. We demonstrate that the HMG box of SOX2 binds in vitro with Homo sapiens-specific derived A-allele and ancestral T-allele carrying DNA sites in BE-HAE hs1210. Molecular docking and simulation analysis indicated highly favourable binding of HMG box with derived A-allele containing DNA site when compared to site carrying ancestral T-allele. CONCLUSION: These results suggest that adoptive changes in TF affinity within BE-HAE hs1210 and other HAR enhancers in the evolutionary history of Homo sapiens might. have brought about changes in gene expression patterns and have functional consequences on forebrain formation and evolution. METHODS: The present study employ electrophoretic mobility shift assays (EMSA) and molecular docking and molecular dynamics simulations approaches.

Asunto(s)

RESUMEN

Rationale: Genetic studies suggest that SOX17 (SRY-related HMG-box 17) deficiency increases pulmonary arterial hypertension (PAH) risk. Objectives: On the basis of pathological roles of estrogen and HIF2α (hypoxia-inducible factor 2α) signaling in pulmonary artery endothelial cells (PAECs), we hypothesized that SOX17 is a target of estrogen signaling that promotes mitochondrial function and attenuates PAH development via HIF2α inhibition. Methods: We used metabolic (Seahorse) and promoter luciferase assays in PAECs together with the chronic hypoxia murine model to test the hypothesis. Measurements and Main Results: Sox17 expression was reduced in PAH tissues (rodent models and from patients). Chronic hypoxic pulmonary hypertension was exacerbated by mice with conditional Tie2-Sox17 (Sox17EC-/-) deletion and attenuated by transgenic Tie2-Sox17 overexpression (Sox17Tg). On the basis of untargeted proteomics, metabolism was the top pathway altered by SOX17 deficiency in PAECs. Mechanistically, we found that HIF2α concentrations were increased in the lungs of Sox17EC-/- and reduced in those from Sox17Tg mice. Increased SOX17 promoted oxidative phosphorylation and mitochondrial function in PAECs, which were partly attenuated by HIF2α overexpression. Rat lungs in males displayed higher Sox17 expression versus females, suggesting repression by estrogen signaling. Supporting 16α-hydroxyestrone (16αOHE; a pathologic estrogen metabolite)-mediated repression of SOX17 promoter activity, Sox17Tg mice attenuated 16αOHE-mediated exacerbations of chronic hypoxic pulmonary hypertension. Finally, in adjusted analyses in patients with PAH, we report novel associations between a SOX17 risk variant, rs10103692, and reduced plasma citrate concentrations (n = 1,326). Conclusions: Cumulatively, SOX17 promotes mitochondrial bioenergetics and attenuates PAH, in part, via inhibition of HIF2α. 16αOHE mediates PAH development via downregulation of SOX17, linking sexual dimorphism and SOX17 genetics in PAH.

Asunto(s)

RESUMEN

Nasopharyngeal carcinoma (NPC) is a highly metastatic and invasive malignant tumor that originates in the nasopharynx. The DNA-binding protein WD repeat and HMG-box DNA-binding protein 1 (WDHD1) are highly expressed in a variety of tumours, but its expression and mechanism of action in NPC have not been reported to date. To investigate the involvement of WDHD1 in NPC, we first mined databases for the gene expression profile of NPC. Immunohistochemistry (IHC) was performed on 338 cases of NPC and 112 non-NPC samples to verify the results. We report that the expression of WDHD1 is significantly elevated in NPC. ChIP-seq was used to show that integrin alpha V (ITGAV) and WDHD1 exhibit a significant binding peak in the promoter region of the ITGAV gene. The expression levels of ITGAV and WDHD1 exhibit a significant positive correlation, and IHC was performed to show that ITGAV is highly expressed in NPC. Expression of ITGAV increased after overexpression of WDHD1, suggesting that ITGAV may be a potential target gene of WDHD1. Pathway analysis showed that both genes were closely related to the cell cycle, and flow cytometry was used to further confirm that decreased expression of WDHD1 significantly increased the number of apoptotic cells. In conclusion, our results suggest that expression of WDHD1 is increased in NPC and is likely to be associated with the NPC cell cycle; thus, we propose that WDHD1 may have the potential as a target gene for primary screening and treatment of NPC.

Asunto(s)

RESUMEN

The mhg1 (NCU02695/ada-23) gene encodes the mitochondrial high-mobility group box (HMG-box or HMGB) protein in Neurospora crassa. The mhg1 KO strain (mhg1KO) has mitochondrial DNA (mtDNA) instability and a short lifespan; however, the function of MHG1 remains unclear. To investigate the role of this protein in the maintenance of mtDNA, domain deleted MHG1 proteins were expressed in the mhg1KO strain, and their effects were analyzed. We identified two putative HMG-domains, HMGBI and HMGBII. Although deletion of the HMG-box did not abolish MHG1's mitochondrial localization, the mhg1KO phenotype of a severe growth defect and a high sensitivity to mutagens could not be restored by introduction of HMG-box deleted mhg1 gene into the KO strain. It was indicated that recombinant full-length MHG1, i.e., mitochondrial targeting sequence (MTS) containing protein, did not exhibit explicit DNA binding, whereas the MHG1 protein truncated for the MTS sequence did in vitro by an electrophoretic mobility shift assay. Furthermore, recombinant MHG1 protein lacking MTS and HMG-domains, either HMGBI or HMGBII, had DNA affinity and an altered band shift pattern compared with MTS-truncated MHG1 protein. These results suggest that cleavage of MTS and appropriate DNA binding via HMG-domains are indispensable for maintaining mtDNA in N. crassa.

Asunto(s)

RESUMEN

Background The term triple-negative breast cancer (TNBC) refers to a particular class of aggressive, poorly differentiated neoplasms that show the absence of estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER2) antibodies. SOX10 (SRY-related HMG-box 10) is a nuclear transcription factor that is commonly used to identify cancers of neural origin, but it has recently been linked to TNBC. The purpose of this study is to determine SOX10 expression in TNBC, its association with tumor grade for molecular categorization, and to determine the diagnostic significance of SOX10 in TNBC at the metastatic site in the case of an unknown primary. Methodology SOX10 was used to stain a tissue microarray of 100 patients. According to the tumor grade, SOX10 staining was classified as negative (<1%), patchy (1-10%), focal (10-70%), and diffuse (70-100%). SPSS version 22 (IBM Corp., Armonk, NY, USA) software was used to conduct the statistical analysis. Results The expression of SOX10 regarding positivity and intensity was higher in high-grade tumors than in intermediate-grade tumors (p = 0.001 and p = 0.007, respectively). Conclusions SOX10 is a reliable novel marker for the diagnosis of TNBC and has diagnostic utility in the unknown primary at the metastatic site.

RESUMEN

The liver has a remarkable regenerative capacity with different modes of regeneration according to the type and extent of an injury. It has been reported that mature hepatocytes could transdifferentiate into a cholangiocyte phenotype. Sry HMG box protein 9 (SOX9) is one of the earliest biliary markers that regulate bile duct development. We have found that SOX9-positive biphenotypic hepatocytes appear in severe acute liver injury patients' liver specimens accompanied by an elevation in plasma interleukin-8 levels. In vitro assays revealed that interleukin-8 homologs induce the expression of SOX9 in mature mouse hepatocytes. Here, we describe the methods used to detect SOX9-positive hepatocytes in human liver specimens and to induce SOX9-positive hepatocytes in mature mouse hepatocytes.

Asunto(s)

RESUMEN

In growing eukaryotic cells, nuclear ribosomal (r)RNA synthesis by RNA polymerase (RNAP) I accounts for the vast majority of cellular transcription. This high output is achieved by the presence of multiple copies of rRNA genes in eukaryotic genomes transcribed at a high rate. In contrast to most of the other transcribed genomic loci, actively transcribed rRNA genes are largely devoid of nucleosomes adapting a characteristic "open" chromatin state, whereas a significant fraction of rRNA genes resides in a transcriptionally inactive nucleosomal "closed" chromatin state. Here, we review our current knowledge about the nature of open rRNA gene chromatin and discuss how this state may be established.

Asunto(s)

RESUMEN

The chromatin-associated high mobility group protein N2 (HMGN2) cofactor regulates transcription factor activity through both chromatin and protein interactions. Hmgn2 expression is known to be developmentally regulated, but the post-transcriptional mechanisms that regulate Hmgn2 expression and its precise roles in tooth development remain unclear. Here, we demonstrate that HMGN2 inhibits the activity of multiple transcription factors as a general mechanism to regulate early development. Bimolecular fluorescence complementation, pull-down, and coimmunoprecipitation assays show that HMGN2 interacts with the transcription factor Lef-1 through its HMG-box domain as well as with other early development transcription factors, Dlx2, FoxJ1, and Pitx2. Furthermore, EMSAs demonstrate that HMGN2 binding to Lef-1 inhibits its DNA-binding activity. We found that Pitx2 and Hmgn2 associate with H4K5ac and H3K4me2 chromatin marks in the proximal Dlx2 promoter, demonstrating Hmgn2 association with open chromatin. In addition, we demonstrate that microRNAs (miRs) mir-23a and miR-23b directly target Hmgn2, promoting transcriptional activation at several gene promoters, including the amelogenin promoter. In vivo, we found that decreased Hmgn2 expression correlates with increased miR-23 expression in craniofacial tissues as the murine embryo develops. Finally, we show that ablation of Hmgn2 in mice results in increased amelogenin expression because of increased Pitx2, Dlx2, Lef-1, and FoxJ1 transcriptional activity. Taken together, our results demonstrate both post-transcriptional regulation of Hmgn2 by miR-23a/b and post-translational regulation of gene expression by Hmgn2-transcription factor interactions. We conclude that HMGN2 regulates tooth development through its interaction with multiple transcription factors.

Asunto(s)

RESUMEN

BACKGROUND: Currently, high expression of WD repeat and HMG-box DNA binding protein 1 (WDHD1) has been found in a variety of tumors; but there is no research has been conducted concerning the expression of WDHD1 in laryngeal squamous cell carcinoma (LSCC). Our purpose is to investigate the expression and the latent mechanism of WDHD1 in LSCC. METHODS: Firstly, 9 data sets from the Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), and ArrayExpress were statistically analyzed to explore the expression of WDHD1 in LSCC; immunohistochemistry was performed in 79 LSCC tissues and 44 non-cancer tissues to further verify the result. In addition, the target gene of WDHD1 was predicted and immunohistochemistry was used to detect the expression of the target gene. The potential mechanism of WDHD1 in LSCC was investigated by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses and protein-protein interaction network (PPI). RESULTS: The WDHD1 mRNA was expressed at higher levels in the LSCC tissue than in the normal tissue (SMD=1.90, 95% CI=1.50-2.30); and the results of immunohistochemistry were consistent with the conclusion. Using chip-seq analysis, we found that S-phase kinase-associated protein 2 (Skp2) had a significant binding peak with WDHD1, and the expression of these two genes was significantly positively correlated. Immunohistochemistry showed that Skp2 was also highly expressed in LSCC. In addition, GO and KEGG analysis revealed the WDHD1 positively correlated genes was closely related to cell cycle, and PPI analysis identified 10 hub genes: COL7A1, COL4A2, COL4A1, COL4A6, COL11A1, COL5A2, COL1A1, COL13A1, COL8A1 and COL10A1, which may be critical to the progression of LSCC. CONCLUSIONS: WDHD1 was overexpressed in LSCC tissues. Meanwhile, WDHD1 and its target gene Skp2 for transcriptional regulation may play a role in the progression of LSCC by regulating the cell cycle.

Asunto(s)

RESUMEN

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that certain of the western blot assay data shown in Figs. 1C and 6A were strikingly similar to data appearing in different form in another article written by different authors. Owing to the fact that the contentious data in the above article were already under consideration for publication prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a satisfactory reply. The Editor apologizes to the readership for any inconvenience caused. [the original article was published in Molecular Medicine Reports 13: 2221­2228, 2016; DOI: 10.3892/mmr.2016.4788].

RESUMEN

Calcific aortic valve disease (CAVD) is an important health burden due to its increasing prevalence and lack of available approaches. Osteogenic transdifferentiation of aortic valve interstitial cells (AVICs) contributes to valve calcification. SRY-related HMG-box transcription factor 5 (SOX5) is essential for cartilage development. Whether SOX5 is involved in AVIC calcification has not been determined. This study aimed to explore the role of SOX5 in warfarin-induced AVIC calcification. Immunostaining showed decreased SOX5 in human calcific AV and warfarin induced mouse calcific AV tissues compared with human noncalcific AV and control mouse AV tissues. In calcific human AVICs (hAVICs) and porcine AVICS (pAVICs), both knockdown and overexpression of SOX5 inhibited calcium deposition and osteogenic marker gene expression. Protein expression assays and ChIP assays showed that overexpression of SOX5 led to increased recruitment of SOX5 to the SOX9 promoter and resulted in increased mRNA and protein expression of SOX9. Coimmunoprecipitation and immunofluorescence showed that SOX5 binds to SOX9 with its HMG domain in nucleus. Blue Native PAGE showed overexpression of SOX5 led to multimeric complex formation of SOX5 and resulted in decreased binding of SOX5 to SOX9 similar to the results of knockdown of SOX5. Further ChIP and western blotting assays showed that both knockdown and overexpression of SOX5 resulted in SOX9 initiating transcription of anti-calcific gene LRP6 in warfarin-treated pAVICs. Knockdown of LRP6 rescues the anti-calcification effect of SOX5 overexpression. We found that both loss and gain of function of SOX5 lead to the same phenotype: decreased warfarin induced calcification. The stoichiometry of SOX5 is crucial for cooperation with SOX9, SOX9 nuclear localization and subsequent binding of SOX9 to LRP6 promoter. These results suggest that SOX5 is a potential target for the development of anti-calcification therapy.

Asunto(s)