TSPAN12 regulates retinal vascular development by promoting Norrin- but not Wnt-induced FZD4/beta-catenin signaling.

Mutations in the genes encoding the Wnt receptor Frizzled-4 (FZD4), coreceptor LRP5, or the ligand Norrin disrupt retinal vascular development and cause ophthalmic diseases. Although Norrin is structurally unrelated to Wnts, it binds FZD4 and activates the canonical Wnt pathway. Here we show that the tetraspanin Tspan12 is expressed in the retinal vasculature, and loss of Tspan12 phenocopies defects seen in Fzd4, Lrp5, and Norrin mutant mice. In addition, Tspan12 genetically interacts with Norrin or Lrp5. Overexpressed TSPAN12 associates with the Norrin-receptor complex and significantly increases Norrin/beta-catenin but not Wnt/beta-catenin signaling, whereas Tspan12 siRNA abolishes transcriptional responses to Norrin but not Wnt3A in retinal endothelial cells. Signaling defects caused by Norrin or FZD4 mutations that are predicted to impair receptor multimerization are rescued by overexpression of TSPAN12. Our data indicate that Norrin multimers and TSPAN12 cooperatively promote multimerization of FZD4 and its associated proteins to elicit physiological levels of signaling.

EGFL7 regulates the collective migration of endothelial cells by restricting their spatial distribution.

During sprouting angiogenesis, groups of endothelial cells (ECs) migrate together in units called sprouts. In this study, we demonstrate that the vascular-specific secreted factor EGFL7 regulates the proper spatial organization of ECs within each sprout and influences their collective movement. In the homozygous Egfl7-knockout mice, vascular development is delayed in many organs despite normal EC proliferation, and 50% of the knockout embryos die in utero. ECs in the mutant vasculatures form abnormal aggregates and the vascular basement membrane marker collagen IV is mislocalized, suggesting that ECs fail to recognize the proper spatial position of their neighbors. Although the migratory ability of individual ECs in isolation is not affected by the loss of EGFL7, the aberrant spatial organization of ECs in the mutant tissues decreases their collective movement. Using in vitro and in vivo analyses, we showed that EGFL7 is a component of the interstitial extracellular matrix deposited on the basal sides of sprouts, a location suitable for conveying positional information to neighboring ECs. Taken together, we propose that EGFL7 defines the optimal path of EC movement by assuring the correct positioning of each EC in a nascent sprout.

Vasoactive intestinal peptide upregulates MUC2 intestinal mucin via CREB/ATF1.

VIP exerts a spectrum of effects as a potent anti-inflammatory factor. In addition, VIP increases expression of MUC2, a major intestinal secretory mucin. We therefore investigated the effects of VIP on the promoter activity of the 5'-flanking region of the MUC2 gene. VIP activated MUC2 transcription in human colonic epithelial cells via cAMP signaling to ERK and p38. cAMP/Epac/Rap1/B-Raf signaling was not involved in MUC2 reporter activation. Furthermore, activation of MUC2 transcription was independent of many of the reported downstream effectors of G protein-coupled receptors, such as PKC, Ras, Raf, Src, calcium, and phosphoinositide 3-kinase. VIP induced cAMP response element-binding protein (CREB)/ATF1 phosphorylation, and this was prevented by treatment with inhibitors of either MEK or p38 and by PKA and MSK1 inhibitor H89. CREB/ATF1 and c-Jun were shown to bind to an oligonucleotide encompassing a distal, conserved CREB/AP1 site in the 5'-flanking region of the MUC2 gene, and this cis element was shown to mediate promoter reporter activation by VIP. This study has identified a new, functional cis element within the MUC2 promoter and also a new pathway regulating MUC2 expression, thus providing further insight into the molecular mechanism of VIP action in the colon. These findings are relevant to the normal biology of the colonic mucosa as well as to the development of VIP as a therapeutic agent for treatment of inflammatory bowel disease.

TNF-alpha activates MUC2 transcription via NF-kappaB but inhibits via JNK activation.

The molecular mechanisms responsible for TNF-alpha-mediated MUC2 intestinal mucin up-regulation in HM3 colon adenocarcinoma cells were analyzed using promoter-reporter assays of the 5'-flanking region of the MUC2 gene. Chemical inhibitors, mutant reporter constructs, and EMSA confirmed I-kappaB/NF-kappaB pathway involvement. Wortmannin, LY294002 and dominant negative Akt, as well as dominant negative NF-kappaB-inducing kinase (NIK) inhibited MUC2 reporter transcription, indicating that both phosphatidylinositol-3-OH kinase (PI3K)/Akt signaling pathway and NIK pathways mediate the effects of TNF-alpha. Wortmannin inhibited NF-kappaB binding and transcriptional activity without inhibiting NF-kappaB translocation to the nucleus, indicating that PI3K/Akt signaling activates NF-kappaB transcriptional activity directly. Our results demonstrate that TNF-alpha up-regulates MUC2 in human colon epithelial cells via several signaling pathways, involving both NIK and PI3K/Akt, which converge at the common IKK/I-kappaB/NF-kappaB pathway. TNF-alpha activated JNK, but JNK inhibitor SP600125 and dominant negative cJun consistently activated transcription, revealing a negative role for this signaling pathway. Thus TNF-alpha causes a net up-regulation of MUC2 gene expression in cultured colon cancer cells because NF-kappaB transcriptional activation of this gene is able to counter-balance the suppressive effects of the JNK pathway. However, the existence of this inhibitory JNK pathways suggests a mechanism whereby--in the absence of NF-kappaB activation--TNF-alpha production during inflammation in vivo could actually inhibit MUC2 production, giving rise to the defective mucosal protection which characterizes inflammatory bowel disease.

Mice expressing SV40 T antigen directed by the intestinal trefoil factor promoter develop tumors resembling human small cell carcinoma of the colon.

The colonic epithelium contains three major types of mature cells, namely, absorptive, goblet, and enteroendocrine cells. These cells are maintained by a complex process of cell renewal involving progenitor and stem cells, and colon cancers develop when this process goes awry. Much is known about the genetic and epigenetic changes that occur in cancer; however, little is known as to the specific cell types involved in carcinogenesis. In this study, we expressed the SV40 Tag oncogene in the intestinal epithelium under the control of an intestinal trefoil factor (ITF) promoter. This caused tumor formation in the proximal colon with remarkable efficiency. ITFTag tumors were rapidly growing, multifocal, and invasive. ITFTag tumor cells express synaptophysin and contain dense core secretory granules, markers of neuroendocrine differentiation. The cell type involved in the early steps of ITFTag tumorigenesis was studied by examining partially transformed crypts that contained populations of both normal and dysplastic cells. The dysplastic cell population always expressed both Tag and synaptophysin. Cells expressing Tag alone were never observed; however, normal enteroendocrine cells expressing synaptophysin but not Tag were readily visualized. This suggests that ITFTag tumor cells originate from the enteroendocrine cell lineage following a transforming event that results in Tag expression. ITFTag tumors closely resemble human small cell carcinomas of the colon, suggesting the possibility that these tumors might be derived from the enteroendocrine cell lineage as well.

Initiation of transcription of the MUC3A human intestinal mucin from a TATA-less promoter and comparison with the MUC3B amino terminus.

Human intestinal mucin genes MUC3A and MUC3B are members of a membrane mucin gene family residing at chromosome 7q22. In this paper, we utilized genomic and cDNA cloning to elucidate the sequence of the 5'-region of the MUC3A gene including the gene promoter and the amino terminus coding sequence. Following its 21-residue signal peptide, the amino terminus of the mucin consists of a 233-residue Thr-, Ser-, and Pro-rich nonrepetitive sequence that is contiguous with its hypervariable domain of 375-residue repeats. RNase protection analysis and 5'-GeneRacer PCR indicated that MUC3A gene transcripts initiate from multiple start sites along a region spanning approximately 180 bases. The 5'-flanking region of the gene had promoter activity when fused to a luciferase reporter gene in all of the tested cell lines. This region contained binding sites for several transcription factors, including those implicated in the regulation of intestinal genes, but lacked a cognate TATA box. These features of the gene promoter may enable the gene to be expressed at variable levels in several cell types with different repertoires of transcription factors. We also utilized 5'-GeneRacer PCR to determine the sequence of the 5'-terminus of the MUC3B message. The amino termini of the MUC3A and MUC3B mucins are 91% conserved at the amino acid level. Thus, MUC3A and MUC3B have highly conserved amino and carboxyl termini, suggesting a recent duplication of the entire ancestral gene. It remains to be determined whether other members of the 7q22 membrane mucin gene family have amino-terminal domains similar to MUC3A and MUC3B.

N-glycosylation is required for the surface localization of MUC17 mucin.

The nucleic acid sequence of the human gene, MUC17, indicates that this mucin contains an SEA domain, a transmembrane domain, and putative N-glycosylation sites in the carboxyl terminus. Mucins that possess an SEA domain are usually proteolytically cleaved within that domain to yield two subunits, the smaller of which is associated with the surface membrane. Homogenates of ASPC-1 pancreatic cancer cells showed three main bands of immunoreactivity with alpha-SEA (a polyclonal antibody directed against a site downstream of the postulated cleavage site) after SDS-PAGE and Western blotting (38, 45, and 49 kDa). Experiments utilizing N-glycan specific hydrolases showed that the 38 kDa band contained high mannose glycans whereas the 45 and 49 kDa bands contained complex-type glycans. Only two smaller alpha-SEA reactive bands (30 and 32 kDa) were present after cells had been treated with the N-glycosylation inhibitor tunicamycin. Surface biotinylation studies showed that only the forms possessing complex-type N-glycans were localized to the cell surface. Both tunicamycin and brefeldin A, an inhibitor of protein transport, reduced surface localization. In summary, our results indicate that the surface localization of the smaller subunit of MUC17 is dependent on its N-glycosylation status.

Aberrant expression of MUC3 and MUC4 membrane-associated mucins and sialyl Le(x) antigen in pancreatic intraepithelial neoplasia.

INTRODUCTION: Ductal adenocarcinoma of the pancreas has recently been suggested to arise from histologically identifiable ductal lesions known as pancreatic intraepithelial neoplasia (PanINs). Altered levels and patterns of mucin gene expression have been reported to occur in epithelial cancers. AIM: To examine the pattern of expression of membrane-associated mucins, MUC3 and MUC4, and a mucin-associated carbohydrate tumor antigen, sialyl Le(x), in these precursor lesions and ductal adenocarcinoma of the pancreas. METHODOLOGY: A total of 144 PanIN lesions and 85 cases of ductal adenocarcinoma of the pancreas were examined by using immunohistochemistry and in situ hybridization methods. RESULTS: MUC3 showed a progressive increase in expression in PanINs of increasing dysplasia and was also highly expressed in ductal adenocarcinoma. In contrast, neoexpression of MUC4 and sialyl Le(x) antigen was observed, mainly in PanIN-3 and ductal adenocarcinoma. In addition, a decrease in the expression of MUC3 and MUC4 was correlated with the degree of de-differentiation of the tumor. CONCLUSION: Aberrant expression of membrane mucins MUC3 and MUC4 and of a mucin-associated carbohydrate tumor antigen Sialyl Le(x) in PanINs and adenocarcinoma further supports the progression model for pancreatic adenocarcinoma.