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Regulation of sonic hedgehog expression by integrin ?1 and epidermal growth factor receptor in intestinal epithelium.

ABSTRACT: We previously found that conditional deletion of integrin ?1 in intestinal epithelium of mice caused early postnatal lethality and intestinal phenotypic changes including excessive proliferation and defective differentiation of intestinal epithelium due to loss of Hedgehog expression. Here, we link these defects to the Hedgehog (Hh) signaling pathway and show that loss of integrin ?1 leads to excessive phosphorylation of MEK-1 and increased expression of ErbB receptors, including the epidermal growth factor receptor (EGFR). We show that increased EGFR signaling attenuates Hh abundance and that an EGFR inhibitor rescues conditional ?1 integrin null pups from postnatal lethality. These studies link the loss of Hh expression in the intestinal epithelium of integrin ?1-deficient mice to excessive EGFR/MAPK signaling, and identify a unique mechanism for crosstalk between stromal and epithelial signaling pathways that is critical for intestinal epithelial differentiation and function.

SUBMITTER: Xu C

PROVIDER: S-EPMC4350683 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Regulation of sonic hedgehog expression by integrin β1 and epidermal growth factor receptor in intestinal epithelium.

Xu Changxin C Li Xiufen X Topham Matthew K MK Kuwada Scott K SK

IUBMB life 20141030 10

We previously found that conditional deletion of integrin β1 in intestinal epithelium of mice caused early postnatal lethality and intestinal phenotypic changes including excessive proliferation and defective differentiation of intestinal epithelium due to loss of Hedgehog expression. Here, we link these defects to the Hedgehog (Hh) signaling pathway and show that loss of integrin β1 leads to excessive phosphorylation of MEK-1 and increased expression of ErbB receptors, including the epidermal g ...[more]

PMID: 25355554

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Project description:During palatogenesis, the palatal shelves first grow vertically on either side of the tongue before changing their direction of growth to horizontal. The extracellular matrix (ECM) plays an important role in these dynamic changes in palatal shelf morphology. Tenascin-C (TNC) is an ECM glycoprotein that shows unique expression in the posterior part of the palatal shelf, but little is known about the regulation of TNC expression. Since transforming growth factor-beta-3 (TGF-?3) and sonic hedgehog (SHH) signaling are known to play important roles in palatogenesis, we investigated whether TGF-?3 and SHH are involved in the regulation of TNC expression in the developing palate. TGF-?3 increased the expression of TNC mRNA and protein in primary mouse embryonic palatal mesenchymal cells (MEPM) obtained from palatal mesenchyme dissected at embryonic day 13.5-14.0. Interestingly, immunohistochemistry experiments revealed that TNC expression was diminished in K14-cre;Tgfbr2 fl/fl mice that lack the TGF-? type II receptor in palatal epithelial cells and exhibit cleft soft palate, whereas TNC expression was maintained in Wnt1-cre;Tgfbr2 fl/fl mice that lack the TGF-? type II receptor in palatal mesenchymal cells and exhibit a complete cleft palate. SHH also increased the expression of TNC mRNA and protein in MEPM cells. However, although TGF-?3 up-regulated TNC mRNA and protein expression in O9-1 cells (a cranial neural crest cell line), SHH did not. Furthermore, TGF-? inhibited the expression of osteoblastic differentiation markers (osterix and alkaline phosphatase) and induced the expression of fibroblastic markers (fibronectin and periostin) in O9-1 cells, whereas SHH did not affect the expression of osteoblastic and fibroblastic markers in O9-1 cells. However, immunohistochemistry experiments showed that TNC expression was diminished in the posterior palatal shelves of Shh-/+ ;MFCS4 +/- mice, which have deficient SHH signaling in the posterior palatal epithelium. Taken together, our findings support the proposal that TGF-? and SHH signaling in palatal epithelium co-ordinate the expression of TNC in the posterior palatal mesenchyme through a paracrine mechanism. This signal cascade may work in the later stage of palatogenesis when cranial neural crest cells have differentiated into fibroblast-like cells. The spatiotemporal regulation of ECM-related proteins by TGF-? and SHH signaling may contribute not only to tissue construction but also to cell differentiation or determination along the anterior-posterior axis of the palatal shelves.

Project description:The intraflagellar transport (IFT) machinery is essential for cilia assembly, maintenance, and trans-localization of signaling proteins. The IFT machinery consists of two large multiprotein complexes, one of which is the IFT-B. TTC30A and TTC30B are integral components of this complex and were previously shown to have redundant functions in the context of IFT, preventing the disruption of IFT-B and, thus, having a severe ciliogenesis defect upon loss of one paralog. In this study, we re-analyzed the paralog-specific protein complexes and discovered a potential involvement of TTC30A or TTC30B in ciliary signaling. Specifically, we investigated a TTC30A-specific interaction with protein kinase A catalytic subunit α, a negative regulator of Sonic hedgehog (Shh) signaling. Defects in this ciliary signaling pathway are often correlated to synpolydactyly, which, intriguingly, is also linked to a rare TTC30 variant. For an in-depth analysis of this unique interaction and the influence on Shh, TTC30A or B single- and double-knockout hTERT-RPE1 were employed, as well as rescue cells harboring wildtype TTC30 or the corresponding mutation. We could show that mutant TTC30A inhibits the ciliary localization of Smoothened. This observed effect is independent of Patched1 but associated with a distinct phosphorylated PKA substrate accumulation upon treatment with forskolin. This rather prominent phenotype was attenuated in mutant TTC30B. Mass spectrometry analysis of wildtype versus mutated TTC30A or TTC30B uncovered differences in protein complex patterns and identified an impaired TTC30A-IFT57 interaction as the possible link leading to synpolydactyly. We could observe no impact on cilia assembly, leading to the hypothesis that a slight decrease in IFT-B binding can be compensated, but mild phenotypes, like synpolydactyly, can be induced by subtle signaling changes. Our systematic approach revealed the paralog-specific influence of TTC30A KO and mutated TTC30A on the activity of PRKACA and the uptake of Smoothened into the cilium, resulting in a downregulation of Shh. This downregulation, combined with interactome alterations, suggests a potential mechanism of how mutant TTC30A is linked to synpolydactyly.

Dataset Information

Regulation of sonic hedgehog expression by integrin ?1 and epidermal growth factor receptor in intestinal epithelium.

Publications

Regulation of sonic hedgehog expression by integrin β1 and epidermal growth factor receptor in intestinal epithelium.

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