Gene expression during THP-1 differentiation is influenced by vitamin D3 and not vibrational mechanostimulation.

Abstract

BackgroundIn injury or infection, monocytes migrate into the affected tissues from circulation and differentiate into macrophages which are subsequently involved in the inflammatory responses. Macrophage differentiation and activation have been studied in response to multiple chemokines and cytokines. However, mechanical, and physical stimuli can also influence macrophage differentiation, activation, cytokine production, and phagocytic activity.MethodsIn this study the macrophage differentiation from THP-1 monocytes was assessed upon the stimulation with 1,25-dihydroxyvitamin D3 and 1,000 Hz vibrations, using qPCR for quantification of transcript expression. Vitamin D binds the vitamin D receptor (VDR) and subsequently modulates the expression of a variety of genes in monocytes. The effects of the 1,000 Hz vibrational stimulation, and the combined treatment of vitamin D3 and 1000 Hz vibrations were unknown. The differentiation of macrophages was assessed by looking at transcription of macrophage markers (e.g., CD14, CD36), antigen presenting molecules (e.g., HLA-DRA), transcription factors (e.g., LEF-1, TCF7L2), and mechanosensors (e.g., PIEZO1 and PKD2).ResultsThe results showed that vitamin D3 induced THP-1 macrophage differentiation, which was characterized by upregulation of CD14 and CD36, downregulation of HLA-DRA, upregulation of the PKD2 (TRPP2), and an inverse relationship between TCF7L2 and LEF-1, which were upregulated and downregulated respectively. The 1,000 Hz vibrations were sensed from the cells which upregulated PIEZO1 and TCF3, but they did not induce expression of genes that would indicate macrophage differentiation. The mRNA transcription profile in the cells stimulated with the combined treatment was comparable to that of the cells stimulated by the vitamin only. The 1,000 Hz vibrations slightly weakened the effect of the vitamin for the regulation of CD36 and HLA-DMB in the suspension cells, but without causing changes in the regulation patterns. The only exception was the upregulation of TCF3 in the suspension cells, which was influenced by the vibrations. In the adherent cells, the vitamin D3 cancelled the upregulating effect of the 1,000 Hz vibrations and downregulated TCF3. The vitamin also cancelled the upregulation of PIEZO1 gene by the 1,000 Hz vibrations in the combined treatment.ConclusionThe mechanical stimulation with 1,000 Hz vibrations resulted in upregulation of PIEZO1 in THP-1 cells, but it did not affect the differentiation process which was investigated in this study. Vitamin D3 induced THP-1 macrophage differentiation and could potentially influence M2 polarization as observed by upregulation of CD36 and downregulation of HLA-DRA. In addition, in THP-1 cells undergoing the combined stimulation, the gene expression patterns were influenced by vitamin D3, which also ablated the effect of the mechanical stimulus on PIEZO1 upregulation.