Neofunctionalized gene RTl3 and SOX9 collectively modulate COL2A1 expression in human articular chondrocytes

Abstract

Purpose: Retrotransposon-derived DNA is a known source of genetic variation, but the biological significance of these molecules in chondrocytes, the resident cell type present in cartilage, is still unknown. A mammalian retrotransposon transcript (Mart) RTL3/ZCCHC5/Mart3 was found to be differentially expressed in human OA cartilage vs normal samples but the function of RTL3 in cartilage remained unexplored. The goal of our study was to determine the functional role of the mammalian retrotransposon transcript (Mart) RTL3/ZCCHC5/Mart3 in human chondrocytes. Methods: These studies were approved by the institutional review boards (IRBs) of Northeast Ohio Medical University and Summa Health Systems (Akron, OH) prior to the initiation of the studies. Chondrocytes were isolated by sequential enzymatic digestion of discarded and de-identified cartilage samples from patients undergoing total joint arthroplasty. Total RNA was isolated using Trizol. mRNA expression was assessed via Taqman assays and protein expression by immunoblotting using validated antibodies. Human RTL3 and SOX-9 expression was depleted using validated siRNAs (GE Dharmacon ONTARGETplus) and X-tremeGENE siRNA Transfection Reagent (SigmaAldrich). For ectopic expression, human chondrocytes were transfected with a RTL3 expression plasmid (Origene) using X-tremeGENE HP DNA Transfection Reagent (SigmaAldrich). COL2A1 promoter reporter assays employed a pCOL2A1-luciferase reporter (GeneCopoeia) and the luciferase activity normalized with a Renilla reporter (Promega). Statistical analyses were conducted using student t-tests or one-way analysis of variation (ANOVA) with Tukey’s post-hoc tests. Results: RTL3 contains a nucleic acid binding domain so we first determined if RTL3 was directly involved in regulating the expression of chondrocyte signature genes expression through gain of function and loss of function analyses. Upon RTL3 depletion, COL2A1 mRNA and protein levels were significantly reduced and activity of the COL2A1 promoter reporter was also significantly decreased. Transfection with an RTL3 expression plasmid under the control of the CMV promoter significantly increased the COL2A1 mRNA and protein levels and enhanced COL2A1 promoter reporter activity was observed in a dose-dependent manner. Together,these results indicated that RTL3 has a direct role in regulating chondrocyte expression of COL2A1. To determine a possible mode of action, we determined if RTL3 modulates COL2A1 expression via known COL2A1 regulator SOX-9. SOX-9 depletion reduced expression of COL2A1 as well as COL2A1 promoter reporter activity. When RTL3 was depleted, constitutive expression of SOX-9 mRNA and protein were significantly reduced compared to controls and the activity of the COL2A1 promoter reporter was also inhibited. Conversely, over-expression of RTL3 was able to significantly enhance mRNA and protein levels of SOX-9 in chondrocytes and increase COL2A1 promoter reporter activity in vitro. Interestingly, in chondrocytes with SOX-9 knockdown and RTL3 overexpression, COL2A1 promoter activity was half that of cells expressing endogenous SOX-9. These results indicate RTL3 or SOX9 alone are not sufficient for optimal expression of COL2A1 but both act in concert to to regulate COL2A1 expression in chondrocytes. Conclusions: The mammalian retrotransposon RTL3 has lost the ability to retrotranspose but has retained an open reading frame suggestive of new cellular functions. Here, we utilized loss-of-function and gain-of-function approaches to demonstrate a previously unknown role of the mammalian retrotransposon transcript RTL3 in the regulation of COL2A1 in human chondrocytes in vitro. siRNA-depletion of RTL3 reduced constitutive SOX-9 expression, pCOL2A1 promoter reporter activity and COL2A1 transcription. Forced expression of RTL3 rescued these effects. SOX-9 siRNA-depletion and RTL3 over expression experiments elucidated that expression of both RTL3 and SOX-9 are important for COL2A1 regulation in chondrocytes. The details of this previously unknown mechanism of regulation warrants further study.