Introduction and Objective: Small-molecule DYRK1A inhibitors such as harmine stimulate adult human β-cell proliferation; however, only a subset enhances β-cell identity/function. We sought to define the signaling mechanism underlying this phenotype, determine whether it is a class effect or requires DYRK1A inhibition, and identify the responsible pathway in human β-cells.Methods: Primary human islets and R7T1 β-cells were treated with DYRK1A inhibitors (harmine, 2-2c, 5-IT, INDY etc.). β-cell identity and function were assessed by qPCR, single-cell RNA-seq, proteomics, immunoblotting, and immunostaining. DYRK1A/1B and candidate pathways were perturbed genetically or pharmacologically. PKA signaling was assessed by CREB Ser133 phosphorylation, forskolin, and H89 inhibition. Harmine affinity pull-down was analyzed by proteomics, and thermal shift assays were performed using recombinant PKA subunits.Results: Harmine, 2-2c, and 5-IT selectively induced β-cell identity and function genes (e.g., PDX1, MAFA, NKX6.1, GLUT2, GLP1R), whereas other DYRK1A inhibitors did not, despite comparable proliferative effects. Single-cell RNA-seq revealed β-cell-restricted enrichment. Combined DYRK1A/1B silencing did not affect identity gene expression or harmine-induced differentiation, indicating a second pro-differentiation pathway. Affinity pull-down identified the PKA regulatory subunit PRKAR1A as a harmine interactor. Harmine, but not INDY, increased CREB Ser133 phosphorylation; forskolin phenocopied harmine, H89 blocked gene induction, and PRKAR1A silencing or PRKACA activation mimicked harmine effects. Thermal shift assays did not support direct binding to PKA subunits, consistent with indirect PKA engagement.Conclusion: Harmine engages two beneficial pathways in human β cells: DYRK1A inhibition drives proliferation, whereas PKA activation promotes differentiation and enhanced function. Uncoupling these effects will inform development of dual-acting diabetes therapeutics.
P. Wang: None. K. Kumar: None. H. Liu: None. S. Chen: None. E. Karakose: None. S. Khamrui: None. M.B. Lazarus: None. A. Garcia-Ocana: Advisory Panel; Current; Paulex Bio. R. DeVita: None. A. Stewart: Advisory Panel; Current; PaulexBio.
NIH (P30DK020541, R01DK116873, R01DK116904, R01DK125285, R01DK105015, R01DK129196, R01 GM132129, R01 DK139631, R35GM124838, R35 CA232128, and P01 CA203655)
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