Genetic Over-Expression of Cytokeratin 18 in Bovine Luteal Cells: Effects on Intermediate Filament Formation and FasL-Induced Death.

Abstract

The Fas-Fas ligand (FasL) system is an integral component of luteal regression, yet the intrinsic mechanism(s) sensitizing luteal cells to FasL-induced cell death are unknown. Susceptibility to FasL in some cell types is influenced by cytokeratin-containing intermediate filaments, and within these cells the filaments impart resistance to cell death. In previous work we have observed that steroidogenic cells of the bovine corpus luteum (CL) contain cytokeratin 18 (CK18) filaments and thus might be resistant to FasL-induced cell death. Here, we tested this possibility by infecting primary cultures of bovine luteal cells with a full-length cDNA construct for hCK18 in a GFP-containing adenovirus vector with cytomegalovirus promoter. The cells were obtained from bovine CL in the early (Day 5) and late stages (Days 16-18) post-ovulation and prepared for primary cell culture. Cells were cultured overnight and exposed to culture medium (Control), GFP-containing adenovirus vector (Ad.GFP transduced), or CK18-adenovirus vector, also containing GFP (Ad.CK18 transduced) at an optimized MOI for 1 hr. Subsequently, the cells were incubated an additional 16-24 hr in virus-free media, and then exposed to either vehicle or a cytokine cocktail containing FasL (50ng/mL FasL) for 24 hr to induce cell death. The cells were fixed and prepared for detection of GFP, immunocytochemical detection of CK18, and an assessment of cell death. Overall infection efficiency averaged 75% in cultures from early stage CL; whereas only 40% efficiency was achieved for cultures from late stage CL. Approximately 45% of the cells from early stage CL expressed CK18 filaments prior to transduction; whereas >75% of the cells expressed CK18 following treatment with Ad.CK18. In cells from late stage CL, endogenous expression of CK18 filaments was relatively low (~16% of all cells), but increased to ~40% of the cells following treatment with Ad.CK18. Intriguingly, virtually all transduced cells from early and late stage CL contained abundant CK18 aggregates rather than filamentous structures. These aggregates were not observed in Control or GFP-transduced cells nor did they affect the viability of the cells in CK18-transduced cultures. FasL-induced cell death was similar among Control, Ad.GFP-, and Ad.CK18-transduced cultures (14%, 18%, and 16% death, respectively) regardless of CL stage, indicating aggregation of CK18 in transduced cultures did not prevent FasL-induced death. Aggregation of CK18 also did not influence progesterone secretion by cells of early CL before or after FasL treatment (62 versus 60 ng/ml, respectively), but potentially enhanced progesterone secretion by cells of late stage CL (82 versus 181 ng/ml for control and FasL-treated cultures, respectively). Knowing that intermediate filaments consist of heterodimeric proteins (e.g., CK18 paired with CK8), the aggregation of CK18 following transduction might have resulted from a lack of other available cytokeratins. In conclusion, genetic over-expression of CK18 in bovine luteal cells results in CK18 aggregation rather than filament formation, which fails to impair FasL-induced cell death but may influence progesterone secretion. This project was supported by National Research Initiative Competitive Grant no. 2007-35203-18074 from the USDA Cooperative State Research, Education, and Extension Service. (poster)