Transcription of the Transforming Growth Factor Activating Integrin 8 Subunit Is Regulated by SP3, AP-1, and the p38 Pathway
ABSTRACT Integrin alphavbeta8 is a critical regulator of transforming growth factor beta activation in vasculogenesis during development, immune regulation, and endothelial/epithelial-mesenchymal homeostasis. Recent studies have suggested roles for integrin beta8 in the pathogenesis of chronic obstructive pulmonary disease, brain arteriovenous malformations, and select cancers (Araya, J., Cambier, S., Markovics, J. A., Wolters, P., Jablons, D., Hill, A., Finkbeiner, W., Jones, K., Broaddus, V. C., Sheppard, D., Barzcak, A., Xiao, Y., Erle, D. J., and Nishimura, S. L. (2007) J. Clin. Invest. 117, 3551-3562; Su, H., Kim, H., Pawlikowska, L., Kitamura, H., Shen, F., Cambier, S., Markovics, J., Lawton, M. T., Sidney, S., Bollen, A. W., Kwok, P. Y., Reichardt, L., Young, W. L., Yang, G. Y., and Nishimura, S. L. (2010) Am. J. Pathol. 176, 1018-1027; Culhane, A. C., and Quackenbush, J. (2009) Cancer Res. 69, 7480-7485; Cambier, S., Mu, D. Z., O'Connell, D., Boylen, K., Travis, W., Liu, W. H., Broaddus, V. C., and Nishimura, S. L. (2000) Cancer Res. 60, 7084-7093). Here we report the first identification and characterization of the promoter for ITGB8. We show that a SP binding site and a cyclic AMP response element (CRE) in the ITGB8 core promoter are required for its expression and that Sp1, Sp3, and several AP-1 transcription factors form a complex that binds to these sites in a p38-dependent manner. Furthermore, we demonstrate the requirement for Sp3, ATF-2, and p38 for the transcription and protein expression of integrin beta8. Additionally, reduction of SP3 or inhibition of p38 blocks alphavbeta8-mediated transforming growth factor beta activation. These results place integrin beta8 expression and activity under the control of ubiquitous transcription factors in a stress-activated and pro-inflammatory pathway.
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- "This system may not be sensitive enough to detect the signals of the shifted band, which usually represents only a very small portion of the binding complexes. Detection of signal reduction (instead of the shifted band) using this EMSA assay system was recently reported.(37) Moreover, chromatin immunoprecipitation (ChIP) experiments further confirmed the specific and direct interaction between Runx2 and the putative Col10a1 cis-enhancer containing the tandem-repeat Runx2 sites in MCT cells (Fig. 2C, D). "
ABSTRACT: We have recently shown that a 150-bp Col10a1 distal promoter (-4296 to -4147 bp) is sufficient to direct hypertrophic chondrocyte-specific reporter (LacZ) expression in vivo. More recently, through detailed sequence analysis we identified two putative tandem-repeat Runx2 binding sites within the 3'-end of this 150-bp region (TGTGGG-TGTGGC, -4187 to -4176 bp). Candidate electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation, and transfection studies demonstrate that these putative Runx2 sites bind Runx2 and mediate upregulated Col10a1/reporter activity in vitro. Transgenic studies using the 5'-sequence without Runx2 sites were not able to drive the cell-specific LacZ reporter activity, suggesting the in vivo requirement of the Runx2 sites located in the 3'-end in mediating Col10a1/reporter expression. Indeed, mutating the Runx2 sites in the context of the 150-bp promoter abolishes its capacity to drive hypertrophic chondrocyte-specific reporter expression in transgenic mice. We have also generated multiple transgenic mouse lines using only the 3'-sequence containing the Runx2 sites to drive the LacZ gene. Interestingly, no hypertrophic chondrocyte-specific blue staining was observed in these transgenic mice. Together, our data support that Runx2 directly interacts with murine Col10a1 cis-enhancer. This interaction is required but not sufficient for cell-specific Col10a1 promoter activity in vivo. Additional cooperative/repressive elements within the 5'- or 3'-sequences of this 150-bp promoter are needed to work with Runx2 together to mediate cell-specific Col10a1 expression. Further delineation of these elements/factors has the potential to identify novel therapeutic targets for multiple skeletal disorders, including osteoarthritis, that show abnormal Col10a1 expression and altered chondrocyte maturation.Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 12/2011; 26(12):2899-910. DOI:10.1002/jbmr.504 · 6.59 Impact Factor
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- "ITGB8, with its binding partner αV, is expressed in normal epithelial and neuronal cells in vivo and regulates transforming growth factor β (TGFβ) activation in various events, including cell growth, matrix modeling, epithelial-mesenchymal homeostasis, immune regulation and vasculogenesis . Binding of Sp1, Sp3 and AP-1 transcription factors to its core promoter regulates ITGB8 expression in a p38-dependent manner . TGFβactivation could lead to autocrine and paracrine signaling on cell growth and matrix production, which are important for epithelial cell adhesion and motility . "
ABSTRACT: Epigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, and their role in corneal epithelium. Human LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC) epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P = 0.0004, Mann-Whitney U-test). This was validated by quantitative polymerase chain reaction (qPCR). Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response) and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance). As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs. We found expression of miR-143/145 cluster in human corneal epithelium. Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting.PLoS ONE 06/2011; 6(6):e21249. DOI:10.1371/journal.pone.0021249 · 3.23 Impact Factor
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ABSTRACT: Transforming growth factor beta (TGFβ) controls numerous cellular responses, including proliferation, differentiation, apoptosis and migration. This cytokine is produced by many different cell types and has been implicated in the pathogenesis of many diseases, ranging from autoimmune disorders and infectious diseases to fibrosis and cancer. However, TGFβ is always produced as an inactive complex that must be activated to enable binding to its receptor and subsequent function. Recent evidence highlights a crucial role for members of the integrin receptor family in controlling the activation of TGFβ. These pathways are important in human health and disease, and new insights into the biochemical mechanisms that allow integrins to control TGFβ activation could prove useful in the design of therapeutics.Trends in Biochemical Sciences 01/2011; 36(1):47-54. DOI:10.1016/j.tibs.2010.08.002 · 13.52 Impact Factor