Gene Expression Profiles Reveal Extracellular Matrix and Inflammatory Signaling in Radiation-Induced Premature Differentiation of Human Fibroblast in vitro.

Carsten Herskind,Frank A. Giordano,Frederik Wenz,Ahmad Sami,Carsten Sticht

doi:10.3389/fcell.2021.539893

Abstract

PurposeFibroblasts are considered to play a major role in the development of fibrotic reaction after radiotherapy and premature radiation-induced differentiation has been proposed as a cellular basis. The purpose was to relate gene expression profiles to radiation-induced phenotypic changes of human skin fibroblasts relevant for radiogenic fibrosis.Materials and MethodsExponentially growing or confluent human skin fibroblast strains were irradiated in vitro with 1–3 fractions of 4 Gy X-rays. The differentiated phenotype was detected by cytomorphological scoring and immunofluorescence microscopy. Microarray analysis was performed on Human Genome U133 plus2.0 microarrays (Affymetrix) with JMP Genomics software, and pathway analysis with Reactome R-package. The expression levels and kinetics of selected genes were validated with quantitative real-time PCR (qPCR) and Western blotting.ResultsIrradiation of exponentially growing fibroblast with 1 × 4 Gy resulted in phenotypic differentiation over a 5-day period. This was accompanied by downregulation of cell cycle-related genes and upregulation of collagen and other extracellular matrix (ECM)-related genes. Pathway analysis confirmed inactivation of proliferation and upregulation of ECM- and glycosaminoglycan (GAG)-related pathways. Furthermore, pathways related to inflammatory reactions were upregulated, and potential induction and signaling mechanisms were identified. Fractionated irradiation (3 × 4 Gy) of confluent cultures according to a previously published protocol for predicting the risk of fibrosis after radiotherapy showed similar downregulation but differences in upregulated genes and pathways.ConclusionGene expression profiles after irradiation of exponentially growing cells were related to radiation-induced differentiation and inflammatory reactions, and potential signaling mechanisms. Upregulated pathways by different irradiation protocols may reflect different aspects of the fibrogenic process thus providing a model system for further hypothesis-based studies of radiation-induced fibrogenesis.

Highlights

Fibroblasts are the most abundant cells in connective tissue and are the major source of extracellular matrix (ECM) proteins
The purpose of the present work was to study gene expression profiles in relation to radiation-induced phenotypic changes of human skin fibroblasts relevant for radiogenic fibrosis
A marked change in fibroblast morphology was observed in micrographs after irradiation of fibroblasts in sparse cultures

Summary

Introduction

Fibroblasts are the most abundant cells in connective tissue and are the major source of extracellular matrix (ECM) proteins. Heterogeneity of cell populations is observed in fibroblast cultures from donors of all ages, representing a terminal differentiation lineage with a progenitor compartment with potentially mitotic fibroblasts and a functional compartment with postmitotic but metabolically active cells that can remain functional for many months, if not years (Bayreuther et al, 1988a,b; Bayreuther et al, 1992). Treatment with cytotoxic agents such as the alkylating agent Mitomycin C or ionizing radiation induces premature differentiation terminal to a postmitotic phenotype characterized by an increase in cell size with enlarged or multiple nuclei and increased synthesis of ECM proteins (Rodemann, 1989; Rodemann et al, 1991; Herskind et al, 2000). Radiation-induced differentiation of fibroblasts has been proposed as a cellular basis of radiation-induced fibrosis (Rodemann and Bamberg, 1995; Herskind et al, 1998)

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