The Molecular Mechanism Of Goat Mammary Gland Epithelial Cells Apoptosis Regulated By ΔFosB Through MMP-9

ΔFosB is a naturally occurring truncated form of FosB that lacks the C-terminal 101 aa region of FosB containing the transactivation domain and is formed by alternative splicing of an intron-like sequence of fosB mRNA. The fosB gene products, especially ΔFosB, have been reported to regulate cell proliferation, differentiation, and death. As the largest and most complex member of the matrix metalloproteinases(MMPs) family, MMP-9 has been implicated in the degradation ofextracellular matrix, which is closely related to cell proliferation and apoptosis, cell migration, inflammatory response and breast tissue remodeling. Previous studies have described overexpression c-Fos and c-Jun induced the high expression level of MMP-9 on mRNA level and the protein activity is also increased. STAT5 plays critical roles in the regulation of cell-apoptosis related genes expression, which also participated in multiple signaling pathways. High levels of STAT5 induced the activation of EGF signaling pathway has been found in malignant tumors and correlates with higher circulating levels of MMP-9.The previous research of our laboratory found that STAT5 inhibits GMECs apoptosis. Higher level of MMP-9 was expressed in goat mammary gland with mastitis, and MMP-9 is positively related with the somatic cell count in milk. Bioinformatics analysis revealed a number of transcription factor binding sites have been characterized in the upstream regulatory region of the MMP-9 gene, including those for AP-1. Although both ΔFosB, STAT5 and MMP-9 are play important roal in cell proliferation and apoptosis of the GMECs, the interactions among them in the apoptosis of goat mammary gland epithelial cells(GMECs) are still unknown.Therefore, we studied the apoptosis effect of ΔFosB in GMECs by regulating MMP-9 and the effects on cell proliferation and apoptosis. Using qRT-PCR, Western blot and flow cytometry instrument to analysis the expression of apoptosis related gene and the effects on cell proliferation and apoptosis by transfected ΔFosB recombinant adenovirus, MMP-9 inhibitors(SB-3CT), STAT5 agonist and inhibitor(Pimozide and PRL) and CaCl2. And then reveal the molecular mechanism of ΔFosB by regulating MMP-9, to clarify the molecular mechanism of apoptosis in lactation period on GMECs.The results are as follows:(1) GMECs treated by SB-3CT 24 h significantly suppressed MMP-9, Bcl-2, Bcl-xl and CyclinD1 expression and increased Bax, Bad, P53, caspase-3 and caspase-9 expression and in GMECs. CCK-8 cell viability assays revealed SB-3CT increased cell death in GMECs. Flow cytometry of FITC-AnnexinV/PI staining assays showed an increased number of apoptotic GMECs after the GME cells were treated with SB-3CT, an inhibitor of MMP-9.(2) GMECs transfected with ΔFosB overexpression recombinant adenoviruses Ad-HA-ΔFosB promoted the expression of ΔFosB, MMP-9, Bcl-2, Bcl-xl and CyclinD1 inhibited the expression of Bax, Bad, P53, caspase-3 and caspase-9 in GMECs, suppressed GMECs apoptosis and increased cell proliferation. The results of transfected with ΔFosB suppression recombinant adenoviruses Ad-ΔFosB-572 is opposite.(3) Compared with the treatment by SB-3CT singly, GMECs transfected with ΔFosB overexpression recombinant adenoviruses Ad-HA-ΔFosB increased mRNA levels of MMP-9. MMP-9 expression increased by interfering ΔFosB and the effect was specifically abrogated by ΔFosB overexpression. Notably, treatment with SB-3CT abolished the promotion effect of ΔFosB on MMP-9 in GMECs transfected with Ad-HA-ΔFosB. ΔFosB overexpression protected the GMECs from SB-3CT induced apoptosis and transduction with the Ad-ΔFosB-572 aggravated mammary epithelial cells death. In GME cells, ΔFosB increased Bcl-2 expression and this upregulation was inhibited by SB-3CT.(4) Calcium significantly suppressed Bcl-2, Bcl-xl and CyclinD1 expression, increased Bax, Bad, P53, caspase-3 and caspase-9 expression and decreased the Bcl-2/Bax and Bcl-xl/Bax ratios and in GMECs. Calcium induced a dose-dependent inhibit the GMECs proliferation and increase apoptosis, an effect that was inhibited by ΔFosB overexpression. This effect was specifically abrogated by ΔFosB overexpression.(5) MMP-9 expression was inhibited in PRL-treated GMECs. the expression of Bcl-2, Bcl-xl and CyclinD1 were promoted, the expression of Bax, Bad, P53, caspase-3 and caspase-9 were inhibited in GMECs, suppressed GMECs apoptosis and increased cell proliferation. Pimozide treatment promoted the expression of MMP-9. Moreover, Pimozide induced GMECs apoptosis and restrained cell proliferation in a dose-dependent manner.In summary, our findings demonstrate for the first time ΔFosB increases the expression of MMP-9 and exhibits a significantly high survival and proliferation in GMECs. We also report that ΔFosB can protect the GMECs from calcium induced and SB-3CT induced apoptosis, and induce cell proliferation, respectively. Thus, ΔFosB may be a potential target in mammary epithelial cells apoptosis by regulating the expression of MMP-9.