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SOX9 directly regulates IGFBP-4 in the intestinal epithelium.

ABSTRACT: SOX9 regulates cell lineage specification by directly regulating target genes in a discrete number of tissues, and previous reports have shown cell proliferative and suppressive roles for SOX9. Although SOX9 is expressed in colorectal cancer, only a few direct targets have been identified in intestinal epithelial cells. We previously demonstrated increased proliferation in Sox9-deficient crypts through loss-of-function studies, indicating that SOX9 suppresses cell proliferation. In this study, crypt epithelial cells isolated from Sox9-deficient mice were used to identify potential target genes of SOX9. Insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4), an inhibitor of the IGF/IGF receptor pathway, was significantly downregulated in Sox9-deficient intestinal epithelial cells and adenoma cells of Sox9-deficient ApcMin/+ mice. Immunolocalization experiments revealed that IGFBP-4 colocalized with SOX9 in mouse and human intestinal epithelial cells and in specimens from patients with primary colorectal cancer. Reporter assays and chromatin immunoprecipitation demonstrated direct binding of SOX9 to the IGFBP-4 promoter. Overexpression of SOX9 attenuated cell proliferation, which was restored following treatment with a neutralizing antibody against IGFBP-4. These results suggest that SOX9 regulates cell proliferation, at least in part via IGFBP-4. Furthermore, the antiproliferative effect of SOX9 was confirmed in vivo using Sox9-deficient mice, which showed increased tumor burden when bred with ApcMin/+ mice. Our results demonstrate, for the first time, that SOX9 is a transcriptional regulator of IGFBP-4 and that SOX9-induced activation of IGFBP-4 may be one of the mechanisms by which SOX9 suppresses cell proliferation and progression of colon cancer.

SUBMITTER: Shi Z

PROVIDER: S-EPMC3725691 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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SOX9 directly regulates IGFBP-4 in the intestinal epithelium.

Shi Zhongcheng Z Chiang Chi-I CI Mistretta Toni-Ann TA Major Angela A Mori-Akiyama Yuko Y

American journal of physiology. Gastrointestinal and liver physiology 20130509 1

SOX9 regulates cell lineage specification by directly regulating target genes in a discrete number of tissues, and previous reports have shown cell proliferative and suppressive roles for SOX9. Although SOX9 is expressed in colorectal cancer, only a few direct targets have been identified in intestinal epithelial cells. We previously demonstrated increased proliferation in Sox9-deficient crypts through loss-of-function studies, indicating that SOX9 suppresses cell proliferation. In this study, c ...[more]

PMID: 23660500

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Project description:Insulin-like growth factor binding protein 2 (IGFBP-2) is important for acquisition of normal bone mass in mice; however, the mechanism by which IGFBP-2 functions is not defined. These studies investigated the role of IGFBP-2 in stimulating osteoblast differentiation. MC-3T3 preosteoblasts expressed IGFBP-2, and IGFBP-2 knockdown resulted in a substantial delay in osteoblast differentiation, reduced osteocalcin expression and Alizarin red staining. These findings were replicated in primary calvarial osteoblasts obtained from IGFBP-2(-/-) mice, and addition of IGFBP-2 rescued the differentiation program. In contrast, overexpression of IGFBP-2 accelerated the time course of differentiation as well as increasing the total number of differentiating cells. By day 6, IGFBP-2-overexpressing cells expressed twice as much osteocalcin as control cultures and this difference persisted. To determine the mechanism by which IGFBP-2 functions, the interaction between IGFBP-2 and receptor tyrosine phosphatase ? (RPTP?) was examined. Disruption of this interaction inhibited the ability of IGFBP-2 to stimulate AKT activation and osteoblast differentiation. Knockdown of RPTP? enhanced osteoblast differentiation, whereas overexpression of RPTP? was inhibitory. Adding back IGFBP-2 to RPTP?-overexpressing cells was able to rescue cell differentiation via enhancement of AKT activation. To determine the region of IGFBP-2 that mediated this effect, an IGFBP-2 mutant that contained substitutions of key amino acids in the heparin-binding domain-1 (HBD-1) was prepared. This mutant had a major reduction in its ability to stimulate differentiation of calvarial osteoblasts from IGFBP-2(-/-) mice. Addition of a synthetic peptide that contained the HBD-1 sequence to calvarial osteoblasts from IGFBP-2(-/-) mice rescued differentiation and osteocalcin expression. In summary, the results clearly demonstrate that IGFBP-2 stimulates osteoblast differentiation and that this effect is mediated through its heparin-binding domain-1 interacting with RPTP?. The results suggest that stimulation of differentiation is an important mechanism by which IGFBP-2 regulates the acquisition of normal bone mass in mice.

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SOX9 directly regulates IGFBP-4 in the intestinal epithelium.

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SOX9 directly regulates IGFBP-4 in the intestinal epithelium.

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