## 4 Discussion
However, regulation of _GJA1_ expression in TGCT may involve other members of the SOX family. Indeed, _SOX8_ and _SOX9_, known to regulate _Gja1_ expression in Sertoli cell lines [14], show correlated expressions with that of _GJA1_ in TGCT (\(p<1.04\times 10^{-6}\)). CaMKI is a serine/threonine kinase highly expressed in spermatogonia and GC-1 cells. In addition, CaMKI can activate _Gja1_ expression. However, the cooperation between Jun and Fosl2 to regulate _Gja1_ promoter activity seems to be independent of CaMKI. Indeed, it is rather the cooperation between Jun and Junb or Fosl1 to activate the _Gja1_ promoter that is enhanced by CaMKII. Interestingly, Jun has a potential CaMKI phosphorylation site at S295 in mouse and S292 in human, whereas Fosl2 contains seven potential CaMKI phosphorylation sites in mouse and in human (S16, S17, S19, T149, S308, S309, S310) as reported using GPSv5.0 [39]. Thus, the activity of these AP-1 family members may be regulated by CaMKI and contribute to activate _Gja1_ expression in other cell types. Importantly, the increase in _Gja1_ promoter activation by Jun and Fosl1 in the presence of constitutively activated CaMKI is not relevant for spermatogonia because Fosl1 is not expressed in these cells. However, four potential CaMKI phosphorylation sites (S254,S255, S256, S257) can be identified in mouse Fosil1 using GPSv5.0 [39], suggesting that the regulation of Fosil1 activity by CaMKII, leading to regulation of _Gja1_ expression, may be important in other cell types. In addition, the expression of _FOSL1_ is correlated with that of _GJA1_ in TGCT. CaMKII phosphorylates a variety of substrates, including CREB [40]. Hence, regulation of _Gja1_ expression by CaMKII in GC-1 cells, and possibly TGCT, may involve phosphorylation of CREB1 and its recruitment to a potential CRE regulatory element within the proximal region of the _Gja1_ promoter. Indeed, CREB1 and its co-activator CBP are being recruited to the _Gja1_ promoter in rat ovarian granulosa cells [41]. In addition, CREB1 can also regulate _Gja1_ promoter activity indirectly by activating the expressions of _Jun_[42] and possibly _Fosil2_. However, the confirmation of such a regulatory mechanism of _Gja1_ expression by CaMKII phosphorylation of CREB1 will require further investigation. CaMKII can also participate in the activation of extracellular signal-regulated kinase (ERK) [43]. Interestingly, activated ERK1/2 can phosphorylate Jun and Fosil2, influencing their transcriptional activation capabilities [44; 45; 46; 47]. As suggested by forskolin treatments, activations of adenylate cyclase and of the cAMP/PKA pathway leads to increased _Gja1_ promoter activity, resulting in increased protein levels. Although PKA contributes to increased activation of the _Gja1_ promoter through cooperation between Jun and Junb, such activation does not exceed that by PKA alone. However, forskolin-dependent increase in Gja1 protein levels may be attributed to increased Fosil2 protein levels and activation of other signaling pathways dependent on membrane calcium influx [48; 49]. Although treatment of GC-1 cells with forskolin leads only to increases in Junb and Fosil2 protein levels, the activity of other AP-1 members could be increased via phosphorylation. Moreover, the cAMP/PKA signaling pathway could contribute to the activation and recruitment of a transcription factor complex involved in the activation of the _Gja1_ promoter as suggested by an increase in _Gja1_ expression in response to forskolin. Additionally, the Ras signaling pathway, having AP-1 members among its downstream targets, has been reported to be involved in the activation of the human _GJA1_ promoter in NIH3T3 cells [50] and could be relevant in the regulation of this connexin in TGCT. Using mouse transgenic models, _Gja1_ has been reported to play an essential role during spermatogenesis, especially for the stage of primary spermatocytes differentiation [51].