Intracellular Ca2+ is not essential for SHH signaling but is promoted by Shh ligand in embryonic fibroblasts

Background: Sonic Hedgehog (SHH) signaling pathway controls cell proliferation, differentiation, and organ formation. The changes in CA²⁺ and their regulatory role in the context of activated SHH signaling have not been fully examined.

Results: In this study, we induced activation of the SHH signaling pathway using activators, including Full-length Shh protein (Shh) and N-terminal Shh (ShhN) ligands or a Smoothened agonist (SAG), while concurrently manipulating calcium level by either increasing extracellular CA²⁺ concentration or employing BAPTA to chelate intracellular CA²⁺. We found that the activation of SHH target genes by the three activators was not affected by low intracellular CA²⁺ but inhibited by excess CA²⁺. Although both Shh and SAG activate CA²⁺ influx via the store-operated calcium entry (SOCE), their effects differ in the presence of BAPTA: Shh directly increases intracellular CA²⁺ concentration, whereas the effect of SAG is enhanced by high extracellular CA²⁺.

Conclusions: Excessive extracellular CA²⁺ impairs SHH signal transduction, whereas intracellular CA²⁺ levels have a negligible effect. At normal physiological extracellular CA²⁺ concentrations, the depletion of intracellular CA²⁺ does not affect SHH signal transduction.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12860-026-00567-x.

Ca2+; Embryonic fibroblasts; Ligand; SHH signaling.