Chondrogenesis is a multistep process that is essential for endochondral bone formation. Previous results have indicated a role for -catenin and Wnt signaling in this pathway. Here we show the existence of physical and functional interactions between -catenin and Sox9, a transcription factor that is required in successive steps of chondrogenesis. In vivo, either overexpression of Sox9 or inactivation of -catenin in chondrocytes of mouse embryos produces a similar phenotype of dwarfism with decreased chondrocyte proliferation, delayed hypertrophic chondrocyte differentiation, and endochondral bone formation. Furthermore, either inactivation of Sox9 or stabilization of -catenin in chondrocytes also produces a similar phenotype of severe chondrodysplasia. Sox9 markedly inhibits activation of -catenin-dependent promoters and stimulates degradation of -catenin by the ubiquitination/proteasome pathway. Likewise, Sox9 inhibits -catenin-mediated secondary axis induction in Xenopus embryos. -Catenin physically interacts through its Armadillo repeats with the C-terminal transactivation domain of Sox9. We hypothesize that the inhibitory activity of Sox9 is caused by its ability to compete with Tcf/Lef for binding to -catenin, followed by degradation of -catenin. Our results strongly suggest that chondrogenesis is controlled by interactions between Sox9 and the Wnt/-catenin signaling pathway. Chondrogenesis, an obligatory process in endochondral bone formation, starts with the recruitment of chondrogenic mesenchymal cells into condensations. This is followed by the differentiation of these cells into chondrocytes, which produce cartilage-specific extracellular matrix (ECM) proteins including type II collagen and the proteoglycan aggrecan. Chondrocytes then undergo a unidirectional proliferation to form orderly parallel columns, exit the cell cycle, become prehypertrophic, and then hypertrophic. Sox9, a high-mobility-group (HMGbox) transcription factor, is required at sequential steps in this pathway (Bi et al. 1999(Bi et al. , 2001Akiyama et al. 2002).Both the human disease campomelic dysplasia, which is caused by heterozygous mutations in the Sox9 gene and is due to Sox9 haploinsufficiency, as well as Sox9 heterozygous mutant mice are characterized by a general hypoplasia of endochondral bones (Foster et al. 1994;Wagner et al. 1994). Inactivation of Sox9 in limb buds using the Cre recombinase/loxP recombination system before chondrogenic mesenchymal condensations results in the complete absence of mesenchymal condensations and of subsequent cartilage and bone formation, indicating that Sox9 is needed for an early step in chondrogenesis, that of mesenchymal condensations (Akiyama et al. 2002). A similar conclusion was also reached by analysis of mouse embryo chimeras derived from homozygous Sox9 mutant embryonic stem (ES) cells (Bi et al. 1999). That Sox9 is needed at sequential steps is shown by the severe generalized chondrodysplasia of mouse embryos in which Sox9 is deleted after chondrogenic mesenchymal conde...
A lot of previous studies have recently reported that the gut microbiota influences the development of colorectal cancer (CRC) in Western countries, but the role of the gut microbiota in Chinese population must be investigated fully. The goal of this study was to determine the role of the gut microbiome in the initiation and development of CRC. We collected fecal samples of 206 Chinese individuals: 59 with polyp (group P), 54 with adenoma (group A), 51 with colorectal cancer (group CC), and 42 healthy controls (group HC).16S ribosomal RNA (rRNA) was used to compare the microbiota community structures among healthy controls, patients with polyp, and those with adenoma or colorectal cancer. Our study proved that intestinal flora, as a specific indicator, showed significant differences in its diversity and composition. Sobs, Chao, and Ace indexes of group CC were significantly lower than those of the healthy control group (CC group: Sobs, Chao, and Ace indexes were 217.3 ± 69, 4265.1 ± 80.7, and 268.6 ± 78.1, respectively; HC group: Sobs, Chao, and Ace indexes were 228.8 ± 44.4, 272.9 ± 58.6, and 271.9 ± 57.2, respectively). When compared with the healthy individuals, the species richness and diversity of intestinal flora in patients with colorectal cancer were significantly reduced: PCA and PCoA both revealed that a significant separation in bacterial community composition between the CC group and HC group (with PCA using the first two principal component scores of PC1 14.73% and PC2 10.34% of the explained variance, respectively; PCoA : PC1 = 14%, PC2 = 9%, PC3 = 6%). Wilcox tests was used to analyze differences between the two groups, it reveals that Firmicutes (P=0.000356), Fusobacteria (P=0.000001), Proteobacteria (P=0.000796), Spirochaetes (P=0.013421), Synergistetes (P=0.005642) were phyla with significantly different distributions between cases and controls. The proportion of microorganism composition is varying at different stages of colon cancer development: Bacteroidetes (52.14%) and Firmicutes (35.88%) were enriched in the healthy individuals; on the phylum level, the abundance of Bacteroidetes (52.14%-53.92%-52.46%–47.06%) and Firmicutes (35.88%-29.73%-24.27%–25.36%) is decreasing with the development of health-polyp-adenomas-CRC, and the abundance of Proteobacteria (9.33%-12.31%-16.51%–22.37%) is increasing. PCA and PCOA analysis showed there was no significant (P<0.05) difference in species similarity between precancerous and carcinogenic states. However, the composition of the microflora in patients with precancerous lesions (including patients with adenoma and polyp) was proved to have no significant disparity (P<0.05). Our study provides insights into new angles to dig out potential biomarkers in diagnosis and treatment of colorectal cancer and to provide scientific advice for a healthy lifestyle for the sake of gut microbiota.
We have identified PIAS1 (protein inhibitor of activated STAT-1), -3, -x␣, and -x as SOX9-associated polypeptides using the Gal4-based yeast two-hybrid system and a cDNA library derived from a chondrocytic cell line. These PIAS proteins were shown to interact directly with SOX9 in two-hybrid, co-immunoprecipitation, and electrophoretic mobility shift assays. SOX9 was sumoylated in cotransfection experiments with COS-7 cells using PIAS and SUMO-1 (small ubiquitin-like modifier-1) expression vectors. SOX9 was also sumoylated in vitro by PIAS proteins in the presence of SUMO-1, the SUMOactivating enzyme, and the SUMO-conjugating enzyme. In COS-7 cells, PIAS proteins stimulated the SOX9-dependent transcriptional activity of a Col2a1 promoter-enhancer reporter. This increase in reporter activity was paralleled by an increase in the cellular levels of SOX9. Cotransfection with a SUMO-expressing vector further enhanced the transcriptional activity of this SOX9-dependent Col2a1 reporter in COS-7 cells, and this additional activation was inhibited in the presence of either SUMO-1 mutants or PIAS RING domain mutants or by coexpression of a desumoylation enzyme. Immunofluorescence microscopy of SOX9-transfected COS-7 cells showed that the subnuclear distribution of SOX9 became more diffuse in the presence of PIAS1 and SUMO-1. Our results suggest that, by controlling the cellular concentrations of SOX9, PIAS proteins and sumoylation may be part of a major regulatory system of SOX9 functions.SOX9, a transcription factor of the SRY (sex-determining region, Y chromosome) family, is required for the establishment and differentiation of several cell lineages, including those of chondrocytes, Sertoli cells of male gonads, glial cells of the nervous system, and others. During chondrocyte differentiation, SOX9 is expressed abundantly in mouse chondroprogenitor cells and overtly differentiated chondrocytes (1) and regulates transcription of cartilage-specific extracellular matrix molecules such as collagen types II (2), IX (3), and XI (4) and aggrecan (5). Heterozygous mutations in the Sox9 gene cause campomelic dysplasia, a severe skeletal malformation syndrome characterized by a generalized hypoplasia of endochondral bones. SOX9 inactivation studies in mice indicate that SOX9 has an essential role in several steps of chondrogenic differentiation, including mesenchymal condensations and overt differentiation of chondrocytes (1, 6). In the absence of SOX9, no chondrocyte marker genes are expressed (1), but the precise mechanism of transcriptional activation by SOX9 of cartilage-specific genes is only poorly understood. The cellular concentration of SOX9 must be tightly regulated in vivo given that campomelic dysplasia is due to haploinsufficiency of SOX9 and because a modest increase in SOX9 expression results in dwarfism in mice (7).Members of the SOX9 family of transcription factors contain a high mobility group (HMG) 3 box DNA-binding domain that is at least 50% identical to an equivalent domain in the sex-determining factor SRY...
MicroRNAs are small non-coding RNAs that may also function as oncogenes and tumor-suppressor genes, as the abnormal expression of microRNAs is associated with various human tumors. However, the effect of miR-335 on the lung cancer cells remains unclear. The aim of the paper was to study the expression of miR335 in non-small cell lung cancer (NSCLC) and miR335's relation to the metastasis, invasion, and apoptosis in lung cancer cells A549 and H1299. qRT-PCR was used to identify the miR-335 expression. The effects of miR-335 on cell proliferation, apoptosis, and invasion were further analyzed. Luciferase reporter assay and Western blot were to verify Bcl-w and SP1 as potential major target genes of miR-335. Finally, the effect of Bcl-w on miR-335-induced cell survival was determined. Our results showed that miR-335 expression was significantly lower in NSCLC tissue, which was significantly associated with lymph node metastasis. In contrast to cells in blank and negative control groups, incidence of apoptosis was significantly higher (P < 0.05) and the number of cells migrating through matrigel was significantly lower (P < 0.05) in miR-335 mimics transfected group. Western blot and luciferase reporter assay demonstrated that miR-335 could bind to the putative binding sites in Bcl-w (or SP1) mRNA 3'-untranslated region to visibly lower the expression of Bcl-w (or SP1). The introduction of Bcl-w cDNA without 3'-untranslated region abrogated miR-335-induced cell survival. These results indicated that upregulation of miR-335 can simultaneously suppress the invasiveness and promote apoptosis of lung cancer cell A549 and H1299 by targeting Bcl-w and SP1. Therefore, miR-335 may be a potential therapeutic target in NSCLC treatment.
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