Browsing by Author "Hollenberg, Morley D."

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    The EGF Receptor and HER2 Participate in TNF-α-Dependent MAPK Activation and IL-8 Secretion in Intestinal Epithelial Cells
    (Hindawi Publishing Corporation, 2012-07-24) Jijon, Humberto B.; Buret, Andre; Hirota, Christina L.; Hollenberg, Morley D.; Beck, Paul L.
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    Proteinase-Activated Receptor-1 and Immunomodulatory Effects of a PAR1-Activating Peptide in a Mouse Model of Prostatitis
    (2013-12-29) Stanton, M. Mark; Nelson, Lisa K.; Benediktsson, Hallgrimur; Hollenberg, Morley D.; Buret, Andre G.; Ceri, Howard
    Background. Nonbacterial prostatitis has no established etiology. We hypothesized that proteinase-activated receptor-1 (PAR1) can play a role in prostatitis. We therefore investigated the effects of PAR1 stimulation in the context of a new model of murine nonbacterial prostatitis. Methods. Using a hapten (ethanol-dinitrobenzene sulfonic acid- (DNBS-)) induced prostatitis model with both wild-type and PAR1-null mice, we examined (1) the location of PAR1 in the mouse prostate and (2) the impact of a PAR1-activating peptide (TFLLR-NH2: PAR1-TF) on ethanol-DNBS-induced inflammation. Results. Ethanol-DNBS-induced inflammation was maximal at 2 days. In the tissue, PAR1 was expressed predominantly along the apical acini of prostatic epithelium. Although PAR1-TF on its own did not cause inflammation, its coadministration with ethanol-DNBS reduced all indices of acute prostatitis. Further, PAR1-TF administration doubled the prostatic production of interleukin-10 (IL-10) compared with ethanol-DNBS treatment alone. This enhanced IL-10 was not observed in PAR1-null mice and was not caused by the reverse-sequence receptor-inactive peptide, RLLFT-NH2. Surprisingly, PAR1-TF, also diminished ethanol-DNBS-induced inflammation in PAR1-null mice. Conclusions. PAR1 is expressed in the mouse prostate and its activation by PAR1-TF elicits immunomodulatory effects during ethanol-DNBS-induced prostatitis. However, PAR1-TF also diminishes ethanol-DNBS-induced inflammation via a non-PAR1 mechanism by activating an as-yet unknown receptor.
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    The Role and Regulation of Proteinase Activated Receptors (PARs) in Urothelial Carcinoma
    (2020-01-24) de Lima, Stacy Grace; Hollenberg, Morley D.; Hyndman, Matthew Eric; Zijlstra, Andries; Morris, Don G.; Lewis, John D.; Black, Peter McL.; Riabowol, Karl T.
    Introduction: Urothelial carcinoma (UC) is the most common type of bladder cancer in the North American population. UC is the most expensive cancer to treat per patient owing to the absence of noninvasive diagnostic and prognostic tests complemented by ineffective treatments and high recurrence rates necessitating long-term follow-up. One phenomenon that has been well demonstrated in UC is that the expression of proteinases and their inhibitors changes in the UC tumor microenvironment as the tissue progresses from healthy urothelium to low grade and then to high grade tumors. This information has not advanced clinical care likely due to the complexity of the proteolytic cascade in both healthy and tumor microenvironments. Proteinase activated receptors (PAR) are specific cell surface receptors that integrate complex proteinase signals into downstream cellular signaling pathways that have been shown to promote survival, proliferation, migration and invasion in carcinomas, but have yet to be explored in UC. Working hypothesis: UC cells produce proteinases that drive an oncogenic (pro-migratory and invasive) phenotype through proteinase activated receptor (PAR) 1 and 2 induced cell signaling which changes with grade, stage and prognosis of disease. Methods: The hypothesis was tested in 5 bladder and UC-derived cell lines of increasing aggressiveness (PC3-420-010, RT4, TCCSUP, 5637, and T24) with the postulation cell aggressiveness (i.e. migration and invasion capacity) would correlated with proteinase and PAR expression and signaling sensitivity. In vitro testing included: PAR expression (qPCR, IHC), PAR signaling (calcium and western blotting for MAP kinase), PAR cleavage (using a variety of published techniques using transfected PAR-indicator constructs in the cell of interest as well as “bystander” responding cells), proteinase expression (proteomics) and activity (fluorescent substrates, PAR-cleavage modeling) testing complemented by organotypic urothelium-fibroblast modeling, as well as immunohistochemical profiling of the expression and cleavage status of PAR1 in patient tumour samples. Results: PAR 1, 2 are expressed by most cell lines tested, but PAR 3, and 4 are not. The expression and sensitivity of these receptors was found to be lowest in healthy urothelium and low-grade papilloma-derived cell lines, and highest in UC cells with increased capacity to migrate and invade. All cell lines produce proteinases and inhibitors, but only high-grade-derived cells produced proteinases that could cleave PAR1 in an autocrine manner. Agonism of PAR1 or PAR2 had little effect on proliferation, but enhanced migration, and invasion in the cell lines that already had this capacity. Only PAR1 antagonism not PAR2 was able to inhibit migration and invasion. Interestingly, in an organotypic model there seemed to be little effect on cellular behaviour (i.e. proliferation, migration, invasion) but robust effects on phenotype with PAR1 targeting upregulating cellular markers associated with a basal phenotype. Patient-derived invasive UC tissue was found to express PAR1. Conclusions: UC-produced proteinases act through PAR1 and promote migration and invasion in UC cell lines in vitro but not in an organotypic model which supported PAR agonism in initiating cell differentiation. UC patient tissue was found to express PAR1 but more data will be necessary to understand the implications of this expression.
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    The EGF Receptor and HER2 Participate in TNF-α-Dependent MAPK Activation and IL-8 Secretion in Intestinal Epithelial Cells
    (2012-09-05) Jijon, Humberto B.; Buret, Andre; Hirota, Christina L.; Hollenberg, Morley D.; Beck, Paul L.
    TNF-alpha activates multiple mitogen-activated protein kinase (MAPK) cascades in intestinal epithelial cells (IECs) leading to the secretion of interleukin 8 (IL-8), a neutrophil chemoattractant and an angiogenic factor with tumor promoting properties. As the epidermal growth factor receptor (EGFR) is a known transducer of proliferative signals and a potent activator of MAPKs, we hypothesized that the EGFR participates in TNF-dependent MAPK activation and IL-8 secretion by intestinal epithelial cells (IECs). We show that the EGFR is tyrosine-phosphorylated following treatment of IECs (HT-29 and IEC-6) with TNF-alpha. This requires EGFR autophosphorylation as it was blocked by the EGFR kinase inhibitor AG1478. Autophosphorylation was also inhibited by both a Src-kinase inhibitor and the metalloproteinase inhibitor batimastat. TNF treatment of IECs resulted in the accumulation of soluble TGF-alpha; treatment of IECs with batimastat suppressed TGF-alpha release and immunoneutralization of TGF-alpha resulted in decreased EGFR and ERK phosphorylations. TNF-alpha treatment of IECs resulted in an association between EGFR and HER2 and inhibition of HER2 using a specific inhibitor AG879 in combination with AG1478-suppressed TNF-alpha-dependent ERK phosphorylation and IL-8 release. Downregulation of HER2 via siRNA resulted in a significant decrease in ERK phosphorylation and a 50% reduction in IL-8 secretion.