Regulation of cellular communication network factor 1 by Ras homolog family member A in bovine steroidogenic luteal cells

Abstract

Abstract Development of the corpus luteum (CL) requires the growth of a new capillary network from preexisting vasculature, a process known as angiogenesis. Successful building of this capillary network occurs through a sequence of cellular events—differentiation, proliferation, migration, and adhesion—which are regulated by a suite of angiogenic proteins that includes cellular communication network factor 1 (CCN1). We previously reported that the expression of CCN1 was highest in luteal tissue obtained from the early-cycle, 4-d-old bovine CL (i.e., corpus hemorrhagicum) compared to the mid- and late-cycle CL. In the present study, we treated steroidogenic bovine luteal cells from early-cycle CL with luteinizing hormone (LH), but it had no effect on CCN1 expression. Direct stimulation of the canonical LH pathway with forskolin and dibutyryl-cyclic adenosine monophosphate (cAMP), however, inhibited CCN1 mRNA expression. In endothelial cells, stimulation of Ras homolog family member A (RhoA) induces CCN1 expression, whereas RhoA inactivation inhibits it. Yet, it is unknown if regulation of CCN1 in steroidogenic luteal cells works likewise. We hypothesized that a similar mechanism of CCN1 regulation exists in bovine luteal cells and that thrombin, a known RhoA activator, may be a physiologic trigger for this mechanism in the early-cycle CL. To test this hypothesis, ovaries were collected from lactating dairy cows on days 3 or 4 of the estrous cycle, and corpora lutea were dissected and dissociated. Steroidogenic luteal cells were suspended in defined Ham’s F12 medium, supplemented with insulin/transferrin/selenium and gentamicin, and seeded into 6-well plates. After 24 h, spent medium was replaced with fresh Ham’s F12, and the cells were cultured for 24 to 48 h. Cells were treated for 2 h with defined medium, 10% fetal bovine serum (FBS), thrombin (1, 5, 10 U/mL), or Rho Activator II (0.25, 1, 2 μg/mL). Cells were then lysed for RNA extraction, followed by cDNA generation, and quantitative polymerase chain reaction (qPCR). Thrombin (1, 5, 10 U/mL; n = 3) and Rho Activator II (0.25, 1, 2 μg/mL; n = 6) increased (P < 0.05) CCN1 mRNA expression. In summary, CCN1 in bovine steroidogenic luteal cells was induced by thrombin and appeared to be regulated in a Rho-dependent manner. Future work will elucidate the signaling partners downstream of Rho which leads to CCN1 gene expression.