4-Phenylbutyric Acid Activates an NF-κB - Egr-1 Axis to Control Myoblast Proliferation and ECM Gene Expression Profiles

Abstract Myoblast proliferation and differentiation are tightly controlled by epigenetic mechanisms, yet how clinically used epigenetic modulators influence myogenic cell fate remains incompletely understood. Here, we demonstrate that the histone deacetylase inhibitor and chemical chaperone 4-phenylbutyric acid (4-PBA) selectively promotes myoblast proliferation without inducing differentiation in C2C12 cells. Mechanistically, 4-PBA increases histone H3 acetylation at lysines 18 and 27 via downregulation of HDAC5, resulting in activation of NF-κB p65. Chromatin immunoprecipitation identifies early growth response 1 (Egr-1) as a direct transcriptional target of NF-κB p65. Transcriptomic analyses reveal that Egr-1 regulates extracellular matrix- and myogenesis-associated gene programs, including multiple collagen genes. Consistently, 4-PBA induces a transcriptional signature that significantly overlaps with Egr-1-dependent gene expression. Functional studies further establish that the NF-κB p65 - Egr-1 axis is required for 4-PBA-mediated transcriptional remodeling in proliferating myoblasts. Together, these findings uncover an epigenetic mechanism by which 4-PBA modulates myoblast proliferation through HDAC5-dependent histone acetylation and NF-κB p65 - Egr-1 driven transcriptional programs, providing insight into how epigenetic therapeutics influence skeletal muscle cell behavior. Highlight 4-PBA enhances murine myoblast proliferation independently of differentiation induction. 4-PBA enhances H3K18 and H3K27 acetylation through downregulation of HDAC5. NF-κB p65 activates Egr-1 by directly binding to the Egr-1 promoter region. 4-PBA stimulates the HDAC5 - NF-κB p65 - Egr-1 axis drives extracellular matrix-related gene profiles. Competing Interest Statement The author N.T. is a founder and director of ADDVEMO Inc.. ADDVEMO Inc. was not involved in the study design, data collection, analysis, interpretation of data, the writing of the report, or the decision to submit the article for publication. The author N.T. receives research funding from Otsuka Electronics Co. for this study. The author K.T. declares no competing interests. Footnotes Author affiliations updated. Figure 2 in the previous version was not displayed correctly due to a PDF formatting issue. This version corrects the figure display. No scientific content has been changed.