Pharmacological profiling reveals sensitivity of vitamin D/TGF-β cooperative gene regulation to epigenetic and transcription-associated inhibitors

Synergistic gene regulation by the active vitamin D metabolite, 1,25(OH) 2 D 3 and transforming growth factor β (TGF-β) has been reported for a limited number of target genes. Among these, 5-lipoxygenase (5-LO), a key enzyme in leukotriene biosynthesis and innate immune signaling, represents a well-established example of this cooperative regulation. However, the molecular mechanisms underlying this synergy between vitamin D and TGF-β remain incompletely understood. In this study, we investigated the involvement of chromatin-associated and transcriptional regulators in 1,25(OH) 2 D 3 /TGF-β mediated gene regulation in monocytic cell lines. Pharmacological perturbation revealed sensitivity of the cooperative response to inhibition of epigenetic regulators, including class I histone deacetylases (HDACs), bromodomain-containing proteins (BRDs), as well as molecular chaperone heat shock protein (HSP90) and transcriptional/signaling kinases such as cyclin-dependent kinase (CDK) 9 and mitogen-activated protein kinases (MAPKs). Further analysis demonstrated differential regulation of ALOX5 (5-LO), CD14 (CD14) and CXCL8 (interleukin-8 (IL-8)) upon modulation of these regulatory pathways in the monocytic cell lines MonoMac-6 and THP-1. RNA-Sequencing identified additional genes synergistically regulated by combined 1,25(OH) 2 D 3 /TGF-β stimulation that were enriched for immune-related functions and displayed similar sensitivity to pharmacological disruption of transcriptional and chromatin regulatory mechanisms. Furthermore, VDR knockout experiments demonstrated that intact VDR signaling is required for 1,25(OH) 2 D 3 /TGF-β mediated cooperative gene regulation. These findings reveal a broader set of immune-associated genes regulated by 1,25(OH) 2 D 3 /TGF-β cooperation and identify chromatin and transcriptional regulators as important modulators of this response in monocytic cells.