Influence of TGFBR2, TGFB3, DNMT1, and DNMT3A Knockdowns on CTGF, TGFBR2, and DNMT3A in Neonatal and Adult Human Dermal Fibroblasts Cell Lines.

Katarzyna Tomela,Marzena Gajecka,Barbara Ginter-Matuszewska,Justyna A Karolak,Michal Kabza

doi:10.3390/cimb43010023

Abstract

Dermal fibroblasts are responsible for the production of the extracellular matrix that undergoes significant changes during the skin aging process. These changes are partially controlled by the TGF-β signaling, which regulates tissue homeostasis dependently on several genes, including CTGF and DNA methyltransferases. To investigate the potential differences in the regulation of the TGF-β signaling and related molecular pathways at distinct developmental stages, we silenced the expression of TGFB1, TGFB3, TGFBR2, CTGF, DNMT1, and DNMT3A in the neonatal (HDF-N) and adult (HDF-A) human dermal fibroblasts using the RNAi method. Through Western blot, we analyzed the effects of the knockdowns of these genes on the level of the CTGF, TGFBR2, and DNMT3A proteins in both cell lines. In the in vitro assays, we observed that CTGF level was decreased after knockdown of DNMT1 in HDF-N but not in HDF-A. Similarly, the level of DNMT3A was decreased only in HDF-N after silencing of TGFBR2, TGFB3, or DNMT1. TGFBR2 level was lower in HDF-N after knockdown of TGFB3, DNMT1, or DNMT3A, but it was higher in HDF-A after TGFB1 silencing. The reduction of TGFBR2 after silencing of DNMT3A and vice versa in neonatal cells only suggests the developmental stage-specific interactions between these two genes. However, additional studies are needed to explain the dependencies between analyzed proteins.

Highlights

Dermal fibroblasts are specialized cells within the dermis layer of the skin, responsible for the synthesis and remodeling of extracellular matrix (ECM) proteins
The transcriptome and protein profiles vary between dermal fibroblasts from newborns and older individuals, suggesting different mechanisms of gene activation depending on developmental stages [7,8]
The siRNA-mediated knockdown of TGFB3 and DNMT1 resulted in a 41% and 39% reduction of the DNMT3A protein level in human dermal fibroblasts (HDF)-N cells

Summary

Introduction

Dermal fibroblasts are specialized cells within the dermis layer of the skin, responsible for the synthesis and remodeling of extracellular matrix (ECM) proteins. The function of skin dermis is modulated by its own cell-specific circadian clock, a system that determines gene expression fluctuation based on day and night rhythm [1]. Throughout the lifespan, the processes regulating circadian system undergoe changes leading to decreased circadian function and skin aging [3]. During both wound healing and dermal aging, the ECM structure undergoes significant modifications [4,5]. The transcriptome and protein profiles vary between dermal fibroblasts from newborns and older individuals, suggesting different mechanisms of gene activation depending on developmental stages [7,8]

Methods

Results

Discussion

Conclusion