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

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.

Keywords: Ras homolog family member A; angiogenesis; cellular communication network factor 1; corpus luteum; luteinizing hormone.

Figure 1.

Proposed model for CCN1 regulation in bovine steroidogenic luteal cells. Starting at the top left, PAR1 (thrombin receptor) activation results in downstream RhoA activation. Activation of RhoA stimulates formation of F-actin via multiple mechanisms, which in turn permits nuclear translocation of the transcription factors MRTF and YAP, both of which stimulate CCN1 expression in endothelial and cancer cells. In the nucleus, YAP couples with TEAD, leading to transcriptional upregulation of CCN1. At the top right, the canonical luteinizing hormone (LH) pathway is represented. Binding of LH to luteinizing hormone receptor (LHCGR) frees a Gs protein to activate adenylate cyclase (AC). Activated AC generates cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP). Then cAMP binds to regulatory subunits of protein kinase A (PKA), enabling it to activate numerous targets, e.g., CREB. There is a possible interaction between PKA and RhoGDI, which would inhibit RhoA.

Figure 2.

Temporal expression of CCN1. Dispersed steroidogenic luteal cells from early-cycle bovine CL were grown in defined Ham’s F12 medium. Cells were treated with 10% fetal bovine serum (FBS) for 0, 2, 4, 8, or 24 h (n = 5). CCN1 mRNA was analyzed using quantitative polymerase chain reaction (qPCR) and relative quantification (ΔΔCt) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control gene. Asterisks (** and ***) denote significance (P < 0.01 and P < 0.001, respectively).

Figure 3.

Regulation of CCN1 by LH, forskolin, and db-cAMP. Dispersed steroidogenic luteal cells from early-cycle bovine CL were grown in defined Ham’s F12 medium. Cells were treated with either LH (1, 10, and 100 ng/mL; n = 5), forskolin (10, 25, and 50 μM; n = 5), or dibutyryl- cAMP (0.1, 1, and 2 mM; n = 6) (panels a–c, respectively) for 2 h. CCN1 mRNA was analyzed using quantitative polymerase chain reaction (qPCR) and relative quantification (ΔΔCt) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control gene. Different letters denote significance (P < 0.05).

Figure 4.

Regulation of CCN1 by RhoA and thrombin. Dispersed steroidogenic luteal cells from early-cycle bovine CL were grown in defined Ham’s F12 medium. Cells were treated with either Rho activator II (0.25, 1, and 2 μg/mL, panel a) or thrombin (1, 5, and 10 U/mL, panel b) for 2 h. CCN1 mRNA was analyzed using quantitative polymerase chain reaction (qPCR) and relative quantification (ΔΔCt) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control gene. Different letters denote significance (P < 0.05).