Title

Effect of transforming growth factor-beta1 (TGF-beta1) on the expression of astrocytic nitric oxide synthase-2 (NOS-2)

Date of Completion

January 2007

Keywords

Biology, Molecular|Biology, Neuroscience

Degree

Ph.D.

Abstract

Nitric oxide synthase 2 (NOS-2) is the calcium-independent and inducible isoform of NOS, which acts to catalytically convert L-arginine to nitric oxide (NO) and L-citrulline. Increased NOS-2 expression has been found in the brains of patients with neurological disease. Further, experimental evidence suggests that NOS-2 contributes, via its NO production, to neuropathology. In vitro studies have demonstrated that astrocytic NOS-2 contributes directly to or potentiates injury-associated neuronal cell death highlighting the need to study how NOS-2 is regulated in astrocytes specifically. Like NOS-2, the cytokine transforming growth factor-β1 (TGF-β1) is upregulated in certain neuropathological conditions. Interestingly, TGF-β1 can either attenuate or augment NOS-2 expression with the prevailing effect dependent on the cell-type and experimental paradigm under study. However, whether TGF-β1 regulates NOS-2 expression in astrocytes has not been fully elucidated. Thus, the overall goal of this dissertation was to elucidate the effect of TGF-β1 on astrocytic NOS-2 expression. To do this, a method was developed to effectively eradicate microglia from primary astrocyte monolayers. Using these cultures, TGF-β1 was demonstrated to potentiate astrocytic NOS-2 expression specifically by increasing the pool of astrocytes that expressed NOS-2. This effect was neither stimulus- nor target-specific. Further, although this effect was optimal with an 8-24 hr pre-treatment with TGF-β1, this enhancement was not found to be due to induction of the pro-inflammatory cytokines IL-1β and TNFα. Rather, this effect was demonstrated to be dependent on prolonged ALK5 signaling and Smad3 nuclear translocation. Knowledge gained from the current proposal may be germaine for the future development of efficacious therapies for several neurological diseases.^