Insights into GABA receptor signalling in TM3 Leydig cells

gamma-Aminobutyric acid (GABA) is an emerging signalling molecule in endocrine organs, since it is produced by endocrine cells and acts via GABA(A) receptors in a paracrine/autocrine fashion. Testicular Leydig cells are producers and targets for GABA. These cells express GABA(A) receptor subunits and in the murine Leydig cell line TM3 pharmacological activation leads to increased proliferation. The signalling pathway of GABA in these cells is not known in this study. We therefore attempted to elucidate details of GABA(A) signalling in TM3 and adult mouse Leydig cells using several experimental approaches. TM3 cells not only express GABA(A) receptor subunits, but also bind the GABA agonist {[}H-3] muscimol with a binding affinity in the range reported for other endocrine cells (K-d = 2.740 +/- 0.721 nM). However, they exhibit a low B-max value of 28.08 fmol/mg protein. Typical GABA(A) receptor-associated events, including Cl- currents, changes in resting membrane potential, intracellular Ca2+ or cAMP, were not measurable with the methods employed in TM3 cells, or, as studied in part, in primary mouse Leydig cells. GABA or GABA(A) agonist isoguvacine treatment resulted in increased or decreased levels of several mRNAs, including transcription factors (c-fos, hsf-1, egr-1) and cell cycle-associated genes (Cdk2, cyclin D1). In an attempt to verify the cDNA array results and because egr-1 was recently implied in Leydig cell development, we further studied this factor. RT-PCR and Western blotting confirmed a time-dependent regulation of egr-1 in TM3. In the postnatal testis egr-1 was seen in cytoplasmic and nuclear locations of developing Leydig cells, which bear GABA(A) receptors and correspond well to TM3 cells. Thus, GABA acts via an untypical novel signalling pathway in TM3 cells. Further details of this pathway remain to be elucidated. Copyright (c) 2005 S. Karger AG, Base

The action of the mast cell product tryptase on cyclooxygenase-2 (COX2) and subsequent fibroblast proliferation involves activation of the extracellular signal-regulated kinase isoforms 1 and 2 (erk1/2)

The mast cell product tryptase, via protease-activated receptor 2 (PAR2), induces cyclooxygenase-2 (COX2) and 15-deoxy-prostaglandin J2 (15d-PGJ2) synthesis. 15d-PGJ2, through the nuclear peroxisome proliferator activated receptor gamma (PPARγ), subsequently causes fibroblast proliferation. In this study we attempted to determine initial events of the tryptase/PAR2 signaling pathway leading to COX2 induction and fibroblast proliferation. In human fibroblasts (HFFF2), cDNA array, RT-PCR and Western blotting studies demonstrated that tryptase, but not 15d-PGJ2, up-regulates c-jun, c-fos and COX2 expression, and phosphorylates the extracellular signal-regulated kinase isoforms 1 and 2 (erk1/2). Furthermore, tryptase effects on erk1/2, c-jun, c-fos, COX2 and cell proliferation were prevented by PD98059, an inhibitor of the mitogen-activated protein kinase kinase (MEK). Other kinases [P38, stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JUNK), erk5], intracellular Ca 2+ or cAMP were not affected by tryptase/PAR2. Our study identifies crucial intracellular events leading to induction of COX2 and fibroblast proliferation, i.e. a cornerstone of fibrosis. © 2004 Elsevier Inc. All rights reserved.Fil: Frungieri, Monica Beatriz. Ludwig Maximilians Universitat; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Albrecht, Martín. Ludwig Maximilians Universitat; AlemaniaFil: Raemsch, Romi. Ludwig Maximilians Universitat; AlemaniaFil: Mayerhofer, Artur. Ludwig Maximilians Universitat; Alemani