The muscarinic cholinergic receptor (mAChR) subtypes share high sequence similarity except in their third intracellular loop and COOH terminus, domains thought to be involved in signal transduction. Subtypes M1, M3, and M5 couple mainly through Gα_q/11, and M2 and M4 couple mainly through Gα_i/o. Whether subtypes within each of these two groups differ in their signaling pathways remains to be resolved. This study focused on nuclear signaling pathways leading to activation of the transcription factor, serum response factor (SRF). Genes encoding M1, M2, and M3 were co-expressed in Jurkat T lymphocytes with a reporter gene driven by a mutant serum response element, SRE.L, which responds to SRF activation. We show that only M1 mAChR activated SRF through a pathway involving the small GTPase RhoA, with no response observed for M2 and M3. Transfection of GTPase-deficient Gα subunits (GαQL; constitutively active form) demonstrated that SRF was activated by Gα₁₃QL but only marginally by Gα_qQL and Gα₁₂QL in Jurkat cells. Yet transfection of regulator of G protein-signaling protein, RGS2 and RGS4, which inhibit Gα_q/11 activity, indicated that Gα_q/11 and Ca²⁺ mobilization were required for SRF activation by M1. Calmodulin inhibitors suppressed the M1 and the Gα₁₃QL pathways, acting both upstream and downstream of RhoA. However, calcineurin inhibitors and the tyrosine kinase inhibitor genistein selectively suppressed SRF activation by M1, but not by Gα₁₃QL, indicating the presence of separate pathways. The calmodulin-dependent tyrosine kinase Pyk2 was also activated by M1 but not M3, and Pyk2 appears also to play a role in M1-SRF activation, as judged by experiments with two dominant-negative Pyk2 mutants. These results reveal a novel calmodulin-dependent RhoA-SRF signaling pathway unique to the M1 mAChR subtype.

Glucocorticoid (GC) resistance complicates the treatment of ~10–20% of children with nephrotic syndrome (NS), yet the molecular basis for resistance remains unclear. We used RNAseq analysis and in silico algorithm-based approaches on peripheral blood leukocytes from 12 children both at initial NS presentation and after ~7 weeks of GC therapy to identify a 12-gene panel able to differentiate steroid resistant NS (SRNS) from steroid-sensitive NS (SSNS). Among this panel, subsequent validation and analyses of one biologically relevant candidate, sulfatase 2 (SULF2), in up to a total of 66 children, revealed that both SULF2 leukocyte expression and plasma arylsulfatase activity Post/Pre therapy ratios were greater in SSNS vs. SRNS. However, neither plasma SULF2 endosulfatase activity (measured by VEGF binding activity) nor plasma VEGF levels, distinguished SSNS from SRNS, despite VEGF’s reported role as a downstream mediator of SULF2’s effects in glomeruli. Experimental studies of NS-related injury in both rat glomeruli and cultured podocytes also revealed decreased SULF2 expression, which were partially reversible by GC treatment of podocytes. These findings together suggest that SULF2 levels and activity are associated with GC resistance in NS, and that SULF2 may play a protective role in NS via the modulation of downstream mediators distinct from VEGF.