Shashi Uniyal

EDB fibronectin and angiogenesis -- a novel mechanistic pathway

Extra domain-B containing fibronectin (EDB+ FN), a recently proposed marker of angiogenesis, has been shown to be expressed in a number of human cancers and in ocular neovascularization in patients with proliferative diabetic retinopathy. To gain molecular understanding of the functional significance of EDB+ FN, we have investigated possible regulatory mechanisms of induction and its role in endothelial cell proliferation and angiogenesis. Human vascular endothelial cells were cultured in high levels of glucose, and fibrogenic growth factors, transforming growth factor-β1 (TGF-β1) and endothelin-1 (ET-1). Our results show that high glucose levels, TGF-β1, and ET-1 upregulated EDB+ FN expression. Treatment of cells exposed to high glucose with TGF-β1 neutralizing antibody and ET receptor antagonist prevented high glucose-induced EDB+ FN expression. In order to elucidate the functional significance of EDB+ FN upregulation, cells were subjected to in vitro proliferation and angiogenesis assays following EDB peptide treatment and specific EDB+ FN gene silencing. Our results show that exposure of cells to EDB peptide increased vascular endothelial growth factor (VEGF) expression, endothelial proliferation, and tube formation. Furthermore, specific EDB+ FN gene silencing prevented both basal and high glucose-induced VEGF expression and reduced the proliferative capacity of endothelial cells. In conclusion, these results indicate that EDB+ FN is involved in endothelial cell proliferation and vascular morphogenesis, findings which may provide novel avenues for the development of anti-angiogenic therapies.

Src and FAK mediate cell–matrix adhesion‐dependent activation of met during transformation of breast epithelial cells

Cell–matrix adhesion has been shown to promote activation of the hepatocyte growth factor receptor, Met, in a ligand‐independent manner. This process has been linked to transformation and tumorigenesis in a variety of cancer types. In the present report, we describe a key role of integrin signaling via the Src/FAK axis in the activation of Met in breast epithelial and carcinoma cells. Expression of an activated Src mutant in non‐neoplastic breast epithelial cells or in carcinoma cells was found to increase phosphorylation of Met at regulatory tyrosines in the auto‐activation loop domain, correlating with increased cell spreading and filopodia extensions. Furthermore, phosphorylated Met is complexed with β1 integrins and is co‐localized with vinculin and FAK at focal adhesions in epithelial cells expressing activated Src. Conversely, genetic or pharmacological inhibition of Src abrogates constitutive Met phosphorylation in carcinoma cells or epithelial cells expressing activated Src, and inhibits filopodia formation. Interestingly, Src‐dependent phosphorylation of Met requires cell–matrix adhesion, as well as actin stress fiber assembly. Phosphorylation of FAK by Src is also required for Src‐induced Met phosphorylation, emphasizing the importance of the Src/FAK signaling pathway. However, stimulation of Met phosphorylation by addition of exogenous HGF in epithelial cells is refractory to inhibition of Src family kinases, indicating that HGF‐dependent and Src/integrin‐dependent Met activation occur via distinct mechanisms. Together these findings demonstrate a novel mechanism by which the Src/FAK axis links signals from the integrin adhesion complex to promote Met activation in breast epithelial cells. J. Cell. Biochem. 107: 1168–1181, 2009.

Threshold levels of ERK activation for chemotactic migration differ for NGF and EGF in rat pheochromocytoma PC12 cells

In a previous study, we show that stimulation of chemotaxis in rat pheochromocytoma PC12 cells by nerve growth factor (NGF) and epidermal growth factor (EGF) requires activation of the RAS-ERK signaling pathway. In this study, we compared the threshold levels of ERK activation required for EGF and NGF-stimulated chemotaxis in PC12 cells. The threshold ERK activity required for NGF to stimulate chemotaxis was approximately 30% lower than that for EGF. PD98059 treatment inhibited EGF stimulation of growth and chemotaxis; however, stimulation of chemotaxis required an EGF concentration approximately 10 times higher than for stimulation of PC12 cell growth. Thus, ERK-dependent cellular functions can be differentially elicited by the concentration of EGF. Also, treatment of PC12 cells with the PI3-K inhibitor LY294002 reduced ERK activation by NGF; thus, higher NGF concentrations were required to initiate chemotaxis and to achieve the same maximal chemotactic response seen in untreated PC12 cells. Therefore, the threshold NGF concentration to stimulate chemotaxis could be adjusted by the crosstalk between the ERK and PI3-K pathways, and the contributions of PI3-K and ERK to signal chemotaxis varied with the concentrations of NGF used. In comparison, LY294002 treatment had no effect on ERK activation by EGF, but the chemotactic response was reduced at all the concentrations of EGF tested indicating that NGF and EGF differed in the utilization of ERK and PI3-K to signal chemotaxis in PC12 cells. (Mol Cell Biochem 271: 29–41, 2005)

Integrin α2β1 modulates EGF stimulation of Rho GTPase-dependent morphological changes in adherent human rhabdomyosarcoma RD cells

The ability of cells to undergo shape changes is essential for diverse cellular functions including cell growth, differentiation, and movement. The present study examines how an integration of the function of α2β1 integrin with that of the receptor for epidermal growth factor (EGFR) modulates EGF‐stimulated morphological changes in human rhabdomyosarcoma RD transfectant cells. Upon EGF stimulation, RD transfectant cells that lacked α2β1 integrin expression (RDpF) underwent contraction; in contrast, expression of α2β1 on RD cells (RDX2C2) resulted in transient cell spreading. Integrin α2 cytoplasmic domain played a critical role in the observed α2β1‐mediated conversion from a cell rounding to a cell spreading phenotype. Thus, the expression of an α2 cytoplasmic domain deletion variant (X2C0) or a chimeric α2β1 containing the cytoplasmic domain of α4 (X2C4) or α5 (X2C5), instead of α2, failed to mediate spreading upon EGF stimulation. Using dominant negative (DN) mutants of RhoGTPases, results revealed that RhoA activation was required for both EGF‐stimulated responses of cell rounding and spreading, Cdc42 functioned in the re‐spreading of cells after undergoing EGF‐stimulated contraction, and Rac1 was required in α2β1‐mediated RD cell spreading. Therefore, α2β1 integrin function can switch the Rho GTPase‐dependent cell shape changes in RD cells from an EGF‐stimulated cell contraction to a spreading morphology. Together, results show that integrin α2 cytoplasmic domain plays an indispensable role in the ability of integrin α2β1 to modulate EGF stimulation of Rho‐GTPase‐dependent morphological changes in RD cells.

Tolerance induction by acylated peptides: suppression of EAE in the mouse with palmitoylated PLP peptides.

Treatment of SJL mice either before or after challenge with palmitoylated PLP139-151 (PAL139-151) completely suppressed or considerably reduced both acute and relapsing stages of EAE induced with PLP139-151. In the presence of Pertussis toxin, treatment with PAL139-151 was less effective, but treatment with a mixture of PAL139-151 and PAL178-191, the palmitoylated PLP epitope to which T cell recognition spreads, resulted in almost complete protection. Proliferation of lymphocytes from treated mice were sharply reduced, and adoptive transfer of lymph node lymphocytes from treated mice to naive recipients resulted in the reduction of the acute phase of EAE and in delayed relapses following challenge. The results suggest that treatment with PAL139-151 leads to both anergy and the generation of regulatory cells.

ED-B FIBRONECTIN IN NON–SMALL CELL LUNG CARCINOMA

Fibronectin (FN), a matrix glycoprotein, has been shown to undergo alternative splicing exclusively during organogenesis and tumorigenesis. One such splice variant, extradomain-B (ED-B) FN, is normally absent in normal adult tissues and is proposed to be a marker of tumoral angiogenesis. The present study was aimed at elucidating whether ED-B FN is expressed in non–small cell lung carcinomas and whether such aberrant expression correlates with tumoral angiogenesis. Frozen tissues from 28 non–small cell lung carcinomas (consisting of both squamous cell carcinomas and adenocarcinomas) along with paired normal tissue samples were collected from the tissue bank collection of the Department of Pathology, London Health Sciences Center, Canada. Frozen tissue specimens were subjected to RNA extraction and real time reverse transcriptase–polymerase chain reaction (RT-PCR) with respect to total and ED-B FN isoform expression. In addition, paraffin-embedded tissue sections from the same cases were collected for histological analysis using ED-B FN antibody. Tumor tissues were further stained with CD34 antibody and analyzed semiquantitatively for tumor microvessel density. The results demonstrate up-regulation of ED-B FN mRNA levels in lung tumor tissues as compared to paired normal tissues. Furthermore, ED-B FN expression was localized specifically to tumor cells and was found to correlate with tumor microvessel density. These findings provide evidence of possible involvement of ED-B FN in pulmonary tumoral angiogenesis. Furthermore, ED-B FN may potentially be used as a diagnostic marker and a target for antiangiogenic therapy.

Integrin VLA-6 (α6β1) mediates adhesion of mouse bone marrow-derived mast cells to laminin

The development of mast cells from bone marrow precursors and their function as the mucosal‐ or connective‐tissue‐type mast cell are critically dependent on microenvironmental factors. Extracellular matrix proteins, such as collagen, fibronectin, and laminin, may represent insoluble components of the microcnvironment. Recent studies have describcd multiple isoforms of laminin isolated from different tissues. In the present study, adhesion of mouse bone marrow‐derived mast cells (BMMC) and long‐term mast cell lines to Engclbreth‐Holm‐Swarm (EHS) tumor laminin, rat laminin. human merosin, and human placental laminin was compared. The greatest level of adhesion was found with human laminin as the substrate. By use of a newly prepared mouse VLA‐α6 integrin‐specific mAb (MA6) together with the previously described mAb GoH3, VLA‐6 (α6β1) integrin was found to be expressed and utilized by BMMC and long‐term mast cell lines. VLA‐6 has been described as a major laminin receptor with roles in diverse cell functions including cell growth and differentiation. BMMC have been shown to express a 32/67‐kDa laminin receptor.

Mycobacterium smegmatis Expressing a Chimeric Protein MPT64-Proteolipid Protein (PLP) 139–151 Reorganizes the PLP-Specific T Cell Repertoire Favoring a CD8-Mediated Response and Induces a Relapsing Experimental Autoimmune Encephalomyelitis

We infected SJL mice with a recombinant Mycobacterium smegmatis expressing a chimeric protein containing the self-epitope of proteolipid protein 139–151 (p139) fused to MPT64, a secreted protein of Mycobacterium tuberculosis (rMSp139). Infected mice developed a relapsing experimental autoimmune encephalomyelitis (EAE), showing a prevailing demyelination of the CNS, and disease severity was significantly lower in comparison with the one that follows immunization with p139. rMSp139 was not detected in lymph node or spleen in the course of clinical disease development or in the CNS during relapse. Infection with rMSp139 modified the p139-specific T cell repertoire, recruiting the spontaneous p139-specific repertoire and activating CD4+ T cells carrying the BV4 semiprivate rearrangement. T cells carrying the public BV10 rearrangement that are consistently found in the CNS during flares of disease were not activated by infection with rMSp139 because lymph node APCs infected with rMSp139 selectively fail to present the epitope for which BV10 cells are specific. Simultaneously, rMSp139 expanded p139-specific CD8+ cells more efficiently than immunization with peptide in adjuvant. SJL mice vaccinated against the CDR3 sequence of the BV10 public rearrangement reduced usage of the BV10 cells and displayed reduced symptoms during bouts of EAE. Thus, transient peripheral infection with a CNS-cross–reactive nonpathogenic Mycobacterium induces a relapsing EAE that continues long after clearance of the infectious agent. The composition of the self-reactive repertoire activated determines severity and histology of the resulting disease.

Integrin VLA‐6 (α6β1) is transiently expressed during the development of mouse bone marrow‐derived mast cells

In the present study the involvement of VLA‐6 (α6β1) integrin, a laminin receptor, was characterized during the course of mouse bone marrow‐derived mast cell (BMMC) development. Flow cytometry and immunoprecipitation revealed increases in α6 integrin expression during the first 3 weeks, followed by a decline, such that α6β1 was no longer detectable by week 13. Using RT‐PCR, transcripts for α6A but not the α6B isoform were detected. Results from immunoprecipitation and costaining with β1‐or β4‐specific mAb showed the expression of VLA‐6 (α6β1) and not α6β4 heterodimers. Moreover, the ability of BMMC to interact with laminin correlated with the time period of VLA‐6 expression. However, only 40% of adhesion to laminin was inhibited by blocking mAb for α6, indicating the involvement of additional laminin receptor(s). This is supported by the immunoprecipitation of VLA‐2 integrin, also known to have laminin binding properties. Heterogeneity of VLA‐6 expression was also found in connective tissue‐type mast cells; thus, VLA‐6hi and VLA‐6lo subpopulations of peritoneal mast cells were observed. The heterogeneity of VLA‐6 integrin expression in BMMC and CTMC may be relevant to the concept of mast cell heterogeneity as well as to the ability of mast cell precursors to migrate and complete their course of maturation within tissues.