Impact of a Heterozygous C1RR301P/WT Mutation on Collagen Metabolism and Inflammatory Response in Human Gingival Fibroblasts

Abstract

Periodontal Ehlers-Danlos syndrome arising from heterozygous pathogenic mutation in C1R and/or C1S genes is an autosomal-dominant disorder characterized by early-onset periodontitis. Due to the difficulties in obtaining and culturing the patient-derived gingival fibroblasts, we established a model system by introducing a heterozygous C1R^R301P/WT mutation into human TERT-immortalized gingival fibroblasts (hGFBs) to investigate its specific effects on collagen metabolism and inflammatory responses. A heterozygous C1R^R301P/WT mutation was introduced into hGFBs using engineered prime editing. The functional consequences of this mutation were assessed at cellular, molecular, and enzymatic levels using a variety of techniques, including cell growth analysis, collagen deposition quantification, immunocytochemistry, enzyme-linked immunosorbent assay, and quantitative real-time reverse transcription polymerase chain reaction. The C1R^R301P/WT-mutated hGFBs (mhGFBs) exhibited normal morphology and growth rate compared to wild-type hGFBs. However, mhGFBs displayed upregulated procollagen α1(V), MMP-1, and IL-6 mRNA expression while simultaneously downregulating collagen deposition and C1r protein levels. A modest accumulation of unfolded collagens was observed in mhGFBs. The mhGFBs exhibited a heightened inflammatory response, with a more pronounced increase in MMP-1 and IL-6 mRNA expression compared to TNF-α/IL-1β-stimulated hGFBs. Unlike cytokine-stimulated hGFBs, cytokine-stimulated mhGFB did not increase C1R, C1S, procollagen α1(III), and procollagen α1(V) mRNA expression. Our results suggest that the C1R^R301P/WT mutation specifically disrupts collagen metabolism and inflammatory pathways in hGFBs, highlighting the mutation's role in these processes. While other cellular functions appear largely unaffected, these findings underscore the potential of targeting collagen metabolism and inflammation for therapeutic interventions in pEDS.

Keywords: collagen metabolism; complement component 1r/1s (C1r/C1s); heterozygous C1RR301P/WT-mutated human TERT-immortalized gingival fibroblasts (mhGFBs); periodontal Ehlers–Danlos syndrome.

Figure 1

Representative Sanger sequencing chromatograms showing the edit area of C1R-R301P in one of the alleles in the mhGFB cell model (a). ddPCR showing the fractional abundance of the C1R^R301P/WT-edited allele in three different mhGFB clones (b), representing the heterozygous mutation in mhGFBs. hGFB: hTERT-immortalized gingival fibroblasts; hGFB (WT): hGFB (wild type); mhGFBs: heterozygous C1R^R301P/WT-mutated hGFBs; mhGFB,C10: mhGFB clone 10; mhGFB,C13: mhGFB clone 13; mhGFB,C25: mhGFB clone 25; C1R-ctrl: C1R—control.

Figure 2

Representative photomicrographs of hGFBs and mhGFBs in 2D culture (a), filamentous actin (b), vimentin (c), CD90 (d), and PDGFRα (e) staining. Cell nuclei are stained in blue color with 4′,6-diamidino-2-phenylindole (DAPI).

Figure 3

Cell proliferation (a), cell growth (b), cell size (c), and PDT (d) of the hGFBs and mhGFBs were investigated after cultured under identical conditions in 2D culture. Results are presented as mean ± SD.

Figure 4

The C1R/C1S gene mRNA (a) and C1r/C1s protein (b) expressions, as well as mRNA expression of vimentin, procollagen α1(I) (Col1α1), procollagen α1(III) (Col3α1), procollagen α1(V) (Col5α1), matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinase-1 (TIMP-1) (c) in hGFBs and mhGFBs after they were cultured in 2D culture for 24 h. C1R: complement component 1r; C1S: complement component 1s. Results are presented as mean ± SD. * indicates statistical significance, with p < 0.05.

Figure 5

Representative images of type I collagen (a) and unfolded collagens (b) with immunocytochemical staining of hGFBs and mhGFBs after they were cultured in 2D cultures for 24 h. The fluorescence intensity of type I collagen (c) and unfolded collagens (d) was analyzed. Cell nuclei are stained with DAPI. Results are presented as mean ± SD.

Figure 6

Collagen deposition in mhGFBs and hGFBs after they were cultured with FB medium (control) or treated with 50 ng/mL of TNF-α or 100 pg/mL IL-1β for 24 h in 2D culture. Data are presented as mean ± SD. * indicates statistical significance, with p < 0.05.

Figure 7

The mRNA expressions of C1R, C1S, vimentin, procollagen α1(I) (Col1α1), procollagen α1(III) (Col3α1), procollagen α1(V) (Col5α1), IL-6, MMP-1, and TIMP-1 in hGFBs and mhGFBs after they were cultured with or without 50 ng/mL of TNF-α or 100 pg/mL IL-1β in 2D culture for 24 h. Cultures without cytokine treatment served as controls. Data are presented as mean ± SD. * indicates statistical significance, with p < 0.05.