Abstract
Purpose :
LRRC15 is a transmembrane protein containing the 15-leucine rich repeat. It interacts with extracellular matrix proteins and has been reported to be expressed in tumors by activated cancer-associated fibroblasts (CAF). Activated CAFs have a myofibroblast phenotype that share many similarities to myofibroblasts observed in wounds. Here, we show for the first time that LRRC15 is expressed by corneal stromal myofibroblasts both in vitro and in vivo.
Methods :
Proteomics studies were carried out using a human corneal fibroblast cell line (HTK). HTKs were cultured for 6 days with 5 ng/ml TGFβ to induce myofibroblast transformation or with serum free (SF) basal media as a control. Protein was then collected and analyzed with mass spectrometry for protein expression. In separate studies, bulk RNA-seq of cultured rabbit corneal keratocytes (NRK), and single cell RNA-seq of rabbit corneas following photorefractive keratectomy (PRK) were performed.
Results :
We found LRRC15 protein to be highly expressed in HTKs cultured with TGFβ, but not for those cultured in SF media. Immunofluorescence and Western blot analysis for alpha smooth muscle actin protein (αsma) showed that at time of protein collection, HTKs had undergone myofibroblast transformation. To determine whether LRRC15 expression was specific to HTK cells, bulk RNA-seq of primary cultures of NRK cells was performed. Results demonstrated that the gene encoding LRRC15 protein was highly expressed following culture in TGFβ as compared to SF media. To investigate whether LRRC15 is also expressed in vivo in corneal stromal myofibroblasts, single-cell RNA-seq was analyzed at different time points after PRK (7, 21 and 60 days after injury). The gene encoding LRRC15 protein was highly expressed at 21 days, which correlated temporally with the expression of myofibroblast markers and the development of corneal fibrosis.
Conclusions :
These results demonstrate for the first time that LRRC15 expression is not limited to activated CAFs (myofibroblastic CAFs), but that it is also expressed by corneal myofibroblasts of both human and rabbit origin. In CAFs, LRRC15 protein expression induces β1 integrin activation which leads to the formation of mature focal adhesions and results in the development of strong stress fibers, and blocking or depleting LRRC15 inhibits myofibroblast transformation. We speculate that LRRC15 could also represent a novel target for modulating corneal fibrosis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.