Insulin-like growth factor (IGF) induced proliferation of human lung fibroblasts is enhanced by neurotensin

Fibroblasts are key cells in tissue repair and important contributors to the inflammatory response. Insulin-like growth factors (IGFs) have been shown to participate in growth, in immune responses and in tissue repair where they stimulate cell growth. Neurotensin (NT) has been suggested to participate in inflammation and in tissue repair and is an autocrine or paracrine growth factor for several cancer cell types. Here we show that IGF-induced proliferation of fibroblasts is enhanced by NT in a concentration and type 1 NT-receptor dependent manner. This action of NT was blocked by inhibitors of phospholipase C and protein kinase C but not by inhibitors of phosphoinositide-3-kinase. An inhibitor of MEK 1/2 significantly reduced the proliferative effects of the IGFs but NT's ability to enhance IGF-induced proliferation was not effected. The ability of NT to enhance IGF-induced proliferation did not involve an autocrine factor. These results suggest that interactions between NT and the IGFs may contribute to the regulation of fibroblasts in for example, inflamed or injured tissues.     

Expression and localization of Muc4/sialomucin complex (SMC) in the adult and developing rat intestine: implications for Muc4/SMC function

Muc4/sialomucin complex (SMC) is a high molecular mass heterodimeric membrane mucin, encoded by a single gene, and originally discovered in a highly metastatic ascites rat mammary adenocarcinoma. Subsequent studies have shown that it is a prominent component of many accessible and vulnerable epithelia, including the gastrointestinal tract. Immunoblot and immunofluorescence analyses demonstrated that Muc4/SMC expression in the rat small intestine increases from proximal to distal regions and is located predominantly in cells at the base of the crypts. These cells were postulated to be Paneth cells, based on their location, morphology, and secretory granule content. Immunohistochemistry indicated the presence of Muc4/SMC in these granules. Muc4/SMC expression was higher in the rat colon than small intestine and was abundantly present in colonic goblet cells, but not in goblet cells in the small intestine. Immunohistochemistry also suggested the presence of MUC4 in human colonic goblet cells. Biochemical analyses indicated that rat colonic Muc4/SMC is primarily the soluble form of the membrane mucin. Analyses of Muc4/SMC during development of the rat gastrointestinal tract showed its appearance at embryonic day 14 of the esophagus and at day 15 at the surface of the undifferentiated stratified epithelium at the gastroduodenal junction, then later at cell surfaces in the more distal regions of the differentiated epithelium of the small intestine, culminating in expression as an intracellular form in the crypts of the small intestine at about day 21. Limited expression in the colon was observed during development before birth at cell surfaces, with expression as an intracellular form in the goblet cells arising during the second week after birth. These results suggest that membrane mucin Muc4/SMC serves different functions during development of the intestine in the rat, but is primarily a secreted product in the adult animal.     

Expression, location, and interactions of ErbB2 and its intramembrane ligand Muc4 (sialomucin complex) in rat mammary gland during pregnancy

Muc4 (also called Sialomucin complex) is a heterodimeric glycoprotein complex consisting of a peripheral O-glycosylated subunit ASGP-1 (ascites sialoglycoprotein-1) tightly but non-covalently bound to an N-glycosylated transmembrane subunit ASGP-2. Muc4/SMC can act as an intramembrane ligand for ErbB2 via an EGF-like domain present in the transmembrane subunit. The complex is developmentally regulated in normal rat mammary gland and overexpressed in a number of mammary tumors. Overexpression of Muc4/SMC has been shown to block cell-cell and cell-matrix interactions, protect tumor cells from immune surveillance, promote metastasis, and protect from apoptosis. We have investigated whether Muc4/SMC and ErbB2 are co-expressed and co-localized in normal rat mammary gland and whether Muc4/SMC-ErbB2 complex formation is developmentally regulated in this tissue. Muc4/SMC and ErbB2 have different expression patterns and regulatory mechanisms in the developing rat mammary gland, but both are maximally expressed during late pregnancy and lactation. The two proteins form a complex in lactating mammary gland which is not detected in the virgin gland. Moreover, this complex does not contain ErbB3. ErbB2 is co-localized with Muc4/SMC at the apical surfaces of ductal and alveolar cells in lactating gland; however, another form of ErbB2, recognized by a different antibody, localizes to the basolateral surfaces of these cells. ErbB2 phosphorylated on Tyr 1248 co-localized with Muc4/SMC at the apical surface but not at the basolateral surfaces of these cells. To investigate the function of Muc4 in the mammary gland, transgenic mice were derived using an MMTV-Muc4 construct. Interestingly, mammary gland development in the transgenic mice was aberrant, exhibiting a bifurcated pattern, including invasion down the blood vessel, similar to that exhibited by transgenic mice inappropriately expressing activated ErbB2 in the mammary gland. These data provide further evidence of the ability of Muc4/SMC to interact with ErbB2 and influence its behavior in normal epithelia.     

Non-apoptotic desquamation of cells from corneal epithelium: putative role for Muc4/sialomucin complex in cell release and survival

Muc4/sialomucin complex (SMC), a large heterodimeric mucin composed of an extracellular mucin subunit ASGP-1 and a transmembrane subunit ASGP-2, is present at the rat ocular surface localized mainly to the most superficial layers of the epithelia. To investigate corneal homeostasis and the functions of Muc4/SMC at the ocular surface, we developed a corneal epithelial cell culture system from corneal explants, from which migrating cells formed an epithelial sheet resembling the native epithelium with regard to microanatomy, expression of characteristic markers, cell migration, and Muc4/SMC expression. Cells migrating from the explants expressed smooth muscle actin. Proliferation was detected only on the edge of epithelial sheet in the immature epithelium and throughout the sheet in confluent cultures. Microscopy revealed that the epithelial sheet was formed from four to six layers of cells expressing keratin 3 and Muc4/SMC in forms identical to those expressed at ocular surface in vivo. Electron microscopy showed cells in various morphological states in the process of releasing from the surface of the multilayer (desquamating). Surprisingly, few of these cells showed evidence of apoptosis, either by morphological or DNA fragmentation analyses. These results suggest a new model for desquamation from stratified epithelia, in which desquamation and apoptosis are independent and sequential processes. Desquamating cells also exhibit a high level of Muc4/SMC. Since Muc4/SMC has been shown to be a potent anti-adhesive and a repressor of apoptosis, we propose that it plays a role in the non-apoptotic desquamation process.     

Limited proteolysis and biophysical characterization of the lipovitellin homology region in apolipoprotein B

Apolipoprotein B (apoB) is the essential nonexchangeable protein in chylomicrons and very low-density lipoprotein-derived lipoprotein particles, including low-density lipoprotein (LDL). ApoB has been a key target for cardiovascular research because of its essential role in the assembly, secretion, delivery, and receptor binding of LDL. The three-dimensional structure of apoB has not been determined. However, the N-terminal region of apoB is homologous to the lipid storage protein lipovitellin, which allows the modeling of this region based on the X-ray structure of lipovitellin. The model of the N-terminal 17% of apoB (B17) suggests that, like lipovitellin, B17 consists of an N-terminal beta-barrel domain, a helical domain, and a beta-sheet domain (C-sheet). Here we test the validity of this model by limited proteolysis of B17 and the characterization of individual domains expressed in Escherichia coli and insect cell systems that are consistent with the model and proteolysis data. Circular dichroism studies of the individual domains indicate that they are folded and their secondary structures are in agreement with the model. We find that the helical domain and C-sheet of apoB interact with each other in vitro, suggesting a strong interaction between these two domains, even without a covalent peptide bond linkage. Our data suggest that the three lipovitellin-like domains exist in B17. Furthermore, the domains fold independently with secondary structures and stabilities like those of intact B17.     

The leucine-rich repeat protein LRIG1 is a negative regulator of ErbB family receptor tyrosine kinases

The molecular mechanisms by which mammalian receptor tyrosine kinases are negatively regulated remain largely unexplored. Previous genetic and biochemical studies indicate that Kekkon-1, a transmembrane protein containing leucine-rich repeats and an immunoglobulin-like domain in its extracellular region, acts as a feedback negative regulator of epidermal growth factor (EGF) receptor signaling in Drosophila melanogaster development. Here we tested whether the related human LRIG1 (also called Lig-1) protein can act as a negative regulator of EGF receptor and its relatives, ErbB2, ErbB3, and ErbB4. We observed that in co-transfected 293T cells, LRIG1 forms a complex with each of the ErbB receptors independent of growth factor binding. We further observed that co-expression of LRIG1 with EGF receptor suppresses cellular receptor levels, shortens receptor half-life, and enhances ligand-stimulated receptor ubiquitination. Finally, we observed that co-expression of LRIG1 suppresses EGF-stimulated transformation of NIH3T3 fibroblasts and that the inducible expression of LRIG1 in PC3 prostate tumor cells suppresses EGF- and neuregulin-1-stimulated cell cycle progression. Our observations indicate that LRIG1 is a negative regulator of the ErbB family of receptor tyrosine kinases and suggest that LRIG1-mediated receptor ubiquitination and degradation may contribute to the suppression of ErbB receptor function.     

Regulation of the membrane mucin Muc4 in corneal epithelial cells by proteosomal degradation and TGF‐β

MUC4 is a heterodimeric membrane mucin, composed of a mucin subunit ASGP‐1 (MUC4α) and a transmembrane subunit ASGP‐2 (MUC4β), which has been implicated in the protection of epithelial cell surfaces. In the rat stratified corneal epithelium Muc4 is found predominantly in the most superficial cell layers. Since previous studies in other tissues have shown that Muc4 is regulated by TGF‐β via a proteosomal degradation mechanism, we investigated the regulation of corneal Muc4 in stratified cultures of corneal epithelial cells. Application of proteosome or processing inhibitors led to increases in levels of Muc4, particularly in the basal and intermediate levels of the stratified cultures. These changes were accompanied by increases in Muc4 ubiquitination, chaperone association and incorporation into intracellular aggresomes. In contrast, treatment with TGF‐β resulted in reduced levels of Muc4, which were reversed by proteosome inhibition. The results support a model in which Muc4 precursor is synthesized in all layers of the corneal epithelium, but Muc4 is degraded in basal and intermediate layers by a proteosomal mechanism at least partly dependent on TGF‐β inhibition of Muc4 processing. J. Cell. Physiol. 223: 209–214, 2010. © 2009 Wiley‐Liss, Inc.     

Morphological Changes in the Midgut of Aedes aegypti L. (Diptera: Culicidae) Larvae Following Exposure to an Annona coriacea (Magnoliales: Annonaceae) Extract

Bioinsecticides are important in the control of disease vectors, but data regarding their physiological effects on target insects are incomplete. This study describes morphological changes that occur in the midgut of third instar Aedes aegypti L. (Diptera: Culicidae) following treatment with a methanolic extract of Annona coriacea (Magnoliales: Annonaceae). Dissected midguts were subdivided into anterior and posterior regions and analyzed by light and scanning electron microscopy. Insects exposed to the extract displayed intense, destructive cytoplasmic vacuolization in columnar and regenerative midgut cells. The apical surfaces of columnar cells exhibited cytoplasmic protrusions oriented toward the lumen, suggesting that these cells could be involved in apocrine secretory processes and/or apoptosis. We report that A. coriacea extracts induced morphological alterations in the midgut of A. aegypti midgut larvae, supporting the use of plant extracts for control of the dengue vector.     

Mechanism of activation of the Drosophila EGF Receptor by the TGFalpha ligand Gurken during oogenesis.

We have analyzed the mechanism of activation of the Epidermal growth factor receptor (Egfr) by the transforming growth factor (TGF) alpha-like molecule, Gurken (Grk). Grk is expressed in the oocyte and activates the Egfr in the surrounding follicle cells during oogenesis. We show that expression of either a membrane bound form of Grk (mbGrk), or a secreted form of Grk (secGrk), in either the follicle cells or in the germline, activates the Egfr. In tissue culture cells, both forms can bind to the Egfr; however, only the soluble form can trigger Egfr signaling, which is consistent with the observed cleavage of Grk in vivo. We find that the two transmembrane proteins Star and Brho potentiate the activity of mbGrk. These two proteins collaborate to promote an activating proteolytic cleavage and release of Grk. After cleavage, the extracellular domain of Grk is secreted from the oocyte to activate the Egfr in the follicular epithelium.