Tumor necrosis factor-alpha induces serum amyloid A3 in mouse granulosa cells

TNF-alpha has significant inhibitory effects on steroidogenesis and folliculogenesis and is associated with several inflammatory responses. Because ovulation is an inflammatory reaction, the effects of TNF on the family of acute-phase proteins in granulosa cells were investigated. Granulosa cells from immature mice at 28 d of age were cultured in the presence of 10 ng TNF/ml for 24 h. Serum amyloid A3 (SAA3), a main acute-phase protein, was induced by TNF in granulosa cells. The other isoforms of serum amyloid proteins SAA1, SAA2, and SAA4 were neither expressed in granulosa cells nor induced by TNF. TNF did not induce SAA3 mRNA in granulosa cells from TNF receptor type 1 (TNFR1) knockout mice, although SAA3 mRNA was induced within 3 h after TNF treatment in wild-type cells. Two SAA3 promoters, -617/+73 and -198/+73, were responsive to TNF and to p65, a component of the TNF signaling molecule nuclear factor (NF)-kappaB. The -106/+73 promoter of SAA3 lacking a NF-kappaB-like site was not responsive to TNF or p65. In granulosa cells from TNFR1 knockout mice, the SAA3 promoter (-198/+73) was responsive to transfection with the p65 component of NF-kappaB, but neither TNF treatment nor overexpression of the p50 component of NF-kappaB increased promoter activity. Similar results were observed in the murine ovarian granulosa tumor cell line (OV3121-1). Overexpression of the inhibitor of NF-kappaB (called IkappaB) blocked SAA3 promoter activity induced by TNF and by p65 in OV3121-1 cells. Closer analysis of deletion mutants of the SAA3 promoter revealed the necessity of a NF-kappaB like site for responsiveness to TNF in the OV3121-1 cells. TNF rapidly increased p65 in OV3121-1 nuclei when compared with controls not treated with TNF. TNF also increased phospho-IkB and SAA3 in whole-cell homogenates as determined by Western blots. Thus, TNF likely increased SAA3 promoter activity and protein by activating NF-kappaB signaling via TNFR1 in mouse granulosa cells. SAA3 is a novel gene in granulosa cells with yet unknown functions in the ovary.