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Cilia-related proteins are believed to be involved in a broad range of cellular processes. Retinitis pigmentosa GTPase regulator interacting protein 1-like (RPGRIP1L) is a ciliary protein required for ciliogenesis in many cell types, including epidermal keratinocytes. Here we report that RPGRIP1L is also involved in the maintenance of desmosomal junctions between keratinocytes. Genetically disrupting the Rpgrip1l gene in mice caused intraepidermal blistering, primarily between basal and suprabasal keratinocytes. This blistering phenotype was associated with aberrant expression patterns of desmosomal proteins, impaired desmosome ultrastructure, and compromised cell-cell adhesion in vivo and in vitro. We found that disrupting the RPGRIP1L gene in HaCaT cells, which do not form primary cilia, resulted in mislocalization of desmosomal proteins to the cytoplasm, suggesting a cilia-independent function of RPGRIP1L. Mechanistically, we found that RPGRIP1L regulates the endocytosis of desmogleins such that RPGRIP1L-knockdown not only induced spontaneous desmoglein endocytosis, as determined by AK23 labeling and biotinylation assays, but also exacerbated EGTA- or pemphigus vulgaris IgG-induced desmoglein endocytosis. Accordingly, inhibiting endocytosis with dynasore or sucrose rescued these desmosomal phenotypes. Biotinylation assays on cell surface proteins not only reinforced the role of RPGRIP1L in desmoglein endocytosis, but also suggested that RPGRIP1L may be more broadly involved in endocytosis. Thus, data obtained from this study advanced our understanding of the biological functions of RPGRIP1L by identifying its role in the cellular endocytic pathway., Author summary The desmosome is a type of intercellular junction, essential for cells to adhere to one another. Abnormalities in desmosomes can cause disorders in the hair, skin, and heart, some of which are severe or even fatal. Here, we discovered that RPGRIP1L, a protein known to regulate cilia formation and function, is essential for stabilizing desmosomes of skin keratinocytes. Specifically, suppressing the Rpgrip1l gene in mice or in keratinocytes disrupted the ultrastructure of desmosomes, and compromised cell-cell adhesion in vivo and in vitro. We found that knocking down RPGRIP1L in keratinocytes aberrantly accelerated the internalization of cell membrane desmogleins, key desmosomal cadherins. Inhibiting endocytosis effectively rescued these phenotypes. Biotinylation assays confirmed that desmogleins are likely the primary targets of RPGRIP1L. Interestingly, membrane proteins that are not directly associated with the desmosomes were also found to be internalized in RPGRIP1L-knockdown cells, raising the possibility that RPGRIP1L might regulate endocytosis more broadly. Findings from this study not only identified RPGRIP1L as a regulator of the desmosomes, but also expanded our understanding of cilia-related proteins in the formation of the desmosomal junctions.

Dominant mutations in keratin genes can cause a number of inheritable skin disorders characterized by intraepidermal blistering, epidermal hyperkeratosis, or abnormalities in skin appendages, such as nail plate dystrophy and structural defects in hair. Allele-specific silencing of mutant keratins through RNA interference is a promising therapeutic approach for suppressing the expression of mutant keratins and related phenotypes in the epidermis. However, its effectiveness on skin appendages remains to be confirmed in vivo. In this study, we developed allele-specific small interfering RNAs capable of selectively suppressing the expression of a mutant Krt75 , which causes hair shaft structural defects characterized by the development of blebs along the hair shaft in mice. Hair regenerated from epidermal keratinocyte progenitor cells isolated from mutant Krt75 mouse models reproduced the blebbing phenotype when grafted in vivo. In contrast, mutant cells manipulated with a lentiviral vector expressing mutant Krt75 -specific short hairpin RNA (shRNA) persistently suppressed this phenotype. The phenotypic correction was associated with a significant reduction of mutant Krt75 mRNA in the skin grafts. Thus, data obtained from this study demonstrated the feasibility of utilizing RNA interference to achieve durable correction of hair structural phenotypes through allele-specific silencing of mutant keratin genes.