## 3 Results
### The cAMP/PKA Pathway Activates the Cja1 Promoter in GC-1 Cells  
Since Jun can be activated by PKA [32], we investigated if overexpression of constitutively active PKA enzyme could influence _Gja1_ promoter activity. Interestingly, PKA overexpression alone upregulated the activity of the _Gja1_ promoter by 15.5 folds (Figure 4A). In addition, the activation of the _Gja1_ promoter by PKA was maintained by the cooperation between Jun and Junb in GC-1 cells (Figure 4A). In addition, PKA overexpression increased Sp1-dependent activation of the _Gja1_ promoter (Figure 4B). According to previously published RNA-Seq data (SRA: PRJNA523810 and PRJNA491649) [21; 26], PKA subunits _Prkara2_ and _Prkara_ were highly expressed in spermatogonia (Figure 4C) and GC-1 cells (Figure 4D). Hence, these subunits may participate in the assembly of PKA in spermatogonial cells. Interestingly, the testis-specific _Prkaca_ variant (_Prkaca2_) is highly expressed in spermatocytes as reported previously [33]. Figure 3: CaMK-dependent activation of the _Gja1_ promoter in mouse GC-1 cells. GC-1 cells were co-transfected with an empty expression vector (CTL) or expression vectors for constitutively active forms of CaMKI and CaMKIV along with the mouse _Gja1_ promoter/_Firefly_ luciferase reporter plasmid. (**A**) Increasing concentrations of CaMKI or IV (625 or 1250 ng/mL) are co-transfected with the mouse _Gja1_ promoter/_Firefly_ luciferase reporter plasmid. (**B**) Expression plasmids encoding Jun, Junb or Fosl2 were co-transfected with CaMKI to evaluate their potentials to activate the _Gja1_ promoter/_Firefly_ luciferase reporter plasmid. (**C**) Expression plasmids encoding Sp1 or Sox4 were co-transfected with CaMKI to evaluate their potentials to activate the _Gja1_ promoter/_Firefly_ luciferase reporter plasmid. In (**D**), GC-1 cells were co-transfected with an empty expression vector (CTL) or expression vectors for Jun and other AP-1 members in the absence or presence of an expression vector encoding a constitutively active form of CaMKI along with the mouse _Gja1_ promoter/_Firefly_ luciferase reporter plasmid. The data is presented as fold activations compared to control (CTL) (\(\pm\)SEM). The number of experiments, each performed in triplicate, is indicated (n). Statistics were performed using a two-way ANOVA followed by a Holm-Sidak’s multiple comparison test. Statistically significant differences from control (CTL) are indicated by an asterisk (\({}^{*}p<0.05\)). Since the transcriptional activities of AP-1 members can be modulated by activation of the cAMP/PKA pathway, we evaluated the ability of forskolin, an activator of adenylate cyclase, to regulate \(Gja1\) expression in GC-1 cells. Interestingly, treatments with forskolin 10 \(\upmu\)M for 8 h resulted in the activation of the \(Gja1\) promoter in GC-1 cells (Figure 5A). Use of reporter plasmid pcREB-luc harboring CREB regulatory elements upstream of the _Firefly_ luciferase served as a positive control for activation of the cAMP/PKA pathway by forskolin, leading to phosphorylation of Creb1 in GC-1 cells (Figure 5B). To better assess the effects of forskolin on the regulation of \(Gja1\), GC-1 cells were treated, followed by total protein extractions and quantification of protein levels using Western blots. Consistent with the increase in \(Gja1\) promoter activity in response to forskolin, Gja1 protein levels are also increased under these same treatment conditions (Figure 5C,D). In addition, the protein levels of Junb and Fols2 are increased in response to forskolin (Figure 5C,D), suggesting that the increase of these AP-1 members by forskolin contributes, in part, to the activation of Gja1 expression in GC-1 cells. Figure 4: PKA promotes the activation the \(Gja1\) promoter by AP-1 members in mouse GC-1 cells.