A role for intracellular calcium downstream of G-protein signaling in undifferentiated human embryonic stem cell culture

Abstract

Multiple signalling pathways maintain human embryonic stem cells (hESC) in an undifferentiated state. Here we sought to define the significance of G protein signal transduction in the preservation of this state distinct from other cellular processes. Continuous treatment with drugs targeting G(αs)-, G(α-i/o)- and G(α-q/11)-subunit signalling mediators were assessed in independent hESC lines after 7days to discern effects on normalised alkaline phosphatase positive colony frequency vs total cell content. This identified PLCβ, intracellular free calcium and CAMKII kinase activity downstream of G(α-q/11) as of particular importance to the former. To confirm the significance of this finding we generated an agonist-responsive hESC line transgenic for a G(α-q/11) subunit-coupled receptor and demonstrated that an undifferentiated state could be promoted in the presence of an agonist without exogenously supplied bFGF and that this correlated with elevated intracellular calcium. Similarly, treatment of unmodified hESCs with a range of intracellular free calcium-modulating drugs in biologically defined mTESR culture system lacking exogenous bFGF promoted an hESC phenotype after 1week of continuous culture as defined by co-expression of OCT4 and NANOG. At least one of these drugs, lysophosphatidic acid significantly elevates phosphorylation of calmodulin and STAT3 in this culture system (p<0.05). These findings substantiate a role for G-protein and calcium signalling in undifferentiated hESC culture.

Lysophosphatidic acid stimulates epithelial to mesenchymal transition marker Slug/Snail2 in ovarian cancer cells via Gαi2, Src, and HIF1α signaling nexus

“…SKOV3.ip cells were stimulated with LPA for 20 minutes along with the appropriate vehicle control and the lysates were subjected to the transcription array analysis. Our results indicated that LPA stimulation activated several transcription factors that have previously been shown to be stimulated by LPA including STAT3 [7, 31, 32] and CREB [7, 33, 34], thus establishing the functional validity of our array analysis. In addition, we observed that LPA stimulated the activity of HIF1α by 150-fold compared to the untreated control cells and its activation far exceeded the activation of any other transcription factor on the array (Figure 1A).…”

Coupling switch of P2Y-IP3 receptors mediates differential Ca2+ signaling in human embryonic stem cells and derived cardiovascular progenitor cells

“…They are also involved in the proliferation and cardiac differentiation of mouse (m) ESCs [23,24] and the growth of human adult cardiac progenitors [25]. In addition, G protein-mediated Ca 2+ signals play a role in the maintenance of undifferentiated state of hESCs [26]. ATP has been verified to induce Ca 2+ transients in hESCs and derived cardiomyocytes [27].…”

Calcium signaling in human pluripotent stem cells

“…According to this work, the inhibition of the CAMKII kinase by the specific inhibitor KN-62 resulted in a decrease in alkaline phosphatase positive (AP+) colony number, suggesting a role of CAMKII kinase in maintaining the pluripotent state [68]. Still, the actual calcium signals evoked by LPA or SERCA pump inhibitors in hPSCs have not been demonstrated.…”

“…5, a combined action of selected GPCRs may occur through calcium independent and calcium-dependent pathways as well. Ermakov et al [68] suggested that an elevation of intracellular calcium may have a significant role in the fate of hPSCs. They found that an elevation of intracellular calcium, induced by the GPCR receptor agonist LPA, or by SERCA pump inhibitors (CPA, TG and chlorpromazine or clotrimazole), can substitute FGF2 under Upon its activation, G protein exchanges GDP for GTP and dissociates into GTP-bound G␣ and G␤␥ subunits.…”