Smad3-dependent signaling underlies the TGF-β1-mediated enhancement in astrocytic iNOS expression.
We previously demonstrated that transforming growth factor-beta1 (TGF-beta1), while having no effect alone, enhances nitric oxide (NO) production in primary, purified mouse astrocytes induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma), by recruiting a latent population of astrocytes to respond, thereby enhancing the total number of cells that express Nos2. In this investigation, we evaluated the molecular signaling pathway by which this occurs. We found that purified murine primary astrocytes express mRNA for TGFbetaRII as well as the TGFbetaRI subunit activin-like kinase 5 (ALK5), but not ALK1. Immunofluorescence microscopy confirmed the expression of TGFbetaRII and ALK5 protein in astrocytes. Consistent with ALK5 signaling, Smad3 accumulated in the nucleus of astrocytes as early as 30 min after TGF-beta1 (3 ng/mL) treatment and persisted upto 32 hr after TGF-beta1 administration. Addition of ALK5 inhibitors prevented TGF-beta1-mediated Smad3 nuclear accumulation and NO production when given prior to the Nos2 induction stimuli, but not after. Finally, astrocyte cultures derived from Smad3 null mutant mice did not exhibit a TGF-beta1-mediated increase in iNOS expression. Overall, this data suggests that ALK5 signaling and Smad3 nuclear accumulation is required for optimal enhancement of LPS plus IFNgamma-induced NO production in astrocytes by TGF-beta1.
Hamby
Mary E
ME
Department of Neuroscience, University of Connecticut Health Center, Farmington, Connecticut, 06030, USA.
Hewett
James A
JA
Hewett
Sandra J
SJ
D021581:Active Transport, Cell Nucleus / Q000235:genetics / Q000502:physiology; D000818:Animals; D001253:Astrocytes / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002467:Cell Nucleus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis* / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000502:physiology; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D051900:Smad3 Protein / Q000378:metabolism / Q000502:physiology*; D053773:Transforming Growth Factor beta1 / Q000502:physiology*; D015854:Up-Regulation / Q000235:genetics / Q000502:physiology*
D008070:Lipopolysaccharides; D018125:Receptors, Transforming Growth Factor beta; D011994:Recombinant Proteins; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; C508961:Tgfb1 protein, mouse; D053773:Transforming Growth Factor beta1; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625842:Tgfbr1 protein, mouse