A Role for Aquaporin-5 Variants in Regulation of the Actin Cytoskeleton in Non-Epidermolytic Palmoplantar Keratoderma

Diffuse non-epidermolytic palmoplantar keratoderma (NEPPK [MIM 600231]) is characterized by thickening of the epidermis in palms and soles with a barrier defect. Missense variants within the AQP5 gene, encoding the water channel protein Aquaporin-5 (AQP5), are the underlying genetic cause of this autosomal dominant genodermatosis. AQP5 is highly expressed in sweat glands and localises to the plasma membrane of suprabasal keratinocytes in the palmoplantar epidermis (Blaydon et al., 2013Blaydon D.C. Lind L.K. Plagnol V. Linton K.J. Smith F.J.D. Wilson N.J. et al.Mutations in AQP5, encoding a water channel protein, cause autosomal-dominant diffuse nonepidermolytic palmoplantar keratoderma.American Journal of Human Genetics. 2013; 93: 330-335Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Here, diffuse NEPPK biopsies and patient-derived keratinocyte cell lines, obtained with written, informed consent and approved by the research ethics committees of the National Health Service (08/H1102/73) and Umeå University, were used to investigate the biological role of epidermally-expressed AQP5. Given a previously reported association between AQP5 and actin stress fibre formation in MDCK cells (Jensen et al., 2018Jensen H.H. Holst M.R. Login F.H. Morgen J.J. Nejsum L.N. Ectopic expression of aquaporin-5 in noncancerous epithelial MDCK cells changes cellular morphology and actin fibre formation without inducing epithelial-to-mesenchymal transition.American Journal of Physiology - Cell Physiology. 2018; 314: 654-661Crossref Scopus (15) Google Scholar), the actin cytoskeleton in control and diffuse NEPPK palm skin and cells was investigated using immunofluorescent staining. An increased presence of ventral actin stress fibres was observed in both diffuse NEPPK palm (AQP5Ile177Phe) and patient-derived keratinocytes (AQP5Ile45Ser) compared to controls (Figure 1A-D). Furthermore, there was a decrease in actin stress fibres in shRNA-mediated AQP5 knockdown patient-derived keratinocytes (Figure 1E,F and Supplementary Figure S1), reinforcing the existence of a molecular association between AQP5Ile45Ser variant protein and actin stress fibre formation. Phosphorylated myosin light chain 2 (pMLC2) is associated with orchestrating stress fibre assembly (Totsukawa et al., 2000Totsukawa G. Yamakita Y. Yamashiro S. Hartshorne D.J. Sasaki Y. Matsumura F. Distinct Roles of ROCK (Rho-Kinase) and MLCK in Spatial Regulation of MLC Phosphorylation for Assembly of Stress Fibers and Focal Adhesions in 3T3 Fibroblasts.Journal of Cell Biology. 2000; 150: 797-806Crossref PubMed Scopus (0) Google Scholar). Immunocytochemical analysis revealed increased pMLC2 protein levels in patient-derived keratinocytes compared to controls (Figure 1G,H), suggesting an association between AQP5Ile45Ser expression and exacerbated pMLC2 levels, which could be driving the increase in actin stress fibre formation. To gain further insight into the role of epidermal AQP5, a BioID (proximity-dependent biotin identification) assay (Roux et al., 2012Roux K.J. Kim D.I. Raida M. Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.Journal of Cell Biology. 2012; 196: 801-810Crossref PubMed Scopus (0) Google Scholar) was conducted to identify novel AQP5 interacting proteins in keratinocytes. Among the significant hits (Supplementary Table S1), CD44 was of particular interest given its expression in the epidermis (Yasaka et al., 1995Yasaka N. Furue M. Tamaki K. CD44 Expression in Normal Human Skin and Skin Tumors.The Journal of Dermatology. 1995; 22: 88-94Crossref PubMed Google Scholar, Shatirishvili et al., 2016Shatirishvili M. Burk A.S. Franz C.M. Pace G. Kastilan T. Breuhahn K. et al.Epidermal-specific deletion of CD44 reveals a function in keratinocytes in response to mechanical stress.Cell Death and Disease. 2016; 7: 1-12Crossref Scopus (29) Google Scholar) and its functional involvement with actin remodelling (Yonemura et al., 1998Yonemura S. Hirao M. Doi Y. Takahashi N. Kondo T. Tsukita S. et al.Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2.Journal of Cell Biology. 1998; 140: 885-895Crossref PubMed Scopus (0) Google Scholar, Bourguignon, 2008Bourguignon L.Y.W. Hyaluronan-mediated CD44 activation of RhoGTPase signaling and cytoskeleton function promotes tumor progression.Seminars in Cancer Biology. 2008; 18: 251-259Crossref PubMed Scopus (0) Google Scholar). Confocal microscopy analysis of keratinocytes overexpressing wildtype AQP5 demonstrated co-localization of AQP5 with CD44 at the keratinocyte plasma membrane (Figure 2A). In addition, proximity ligation assay (PLA) revealed signals localized at cell-cell contact sites (Figure 2B), suggesting an interaction between AQP5 and CD44 at the keratinocyte plasma membrane. Next, a AQP5 and CD44 co-stain was conducted in body skin, normal palm and two diffuse NEPPK palm biopsies harbouring AQP5Ile177Phe or AQP5Ala38Glu variants (Figure 2C). As previously reported (Blaydon et al., 2013Blaydon D.C. Lind L.K. Plagnol V. Linton K.J. Smith F.J.D. Wilson N.J. et al.Mutations in AQP5, encoding a water channel protein, cause autosomal-dominant diffuse nonepidermolytic palmoplantar keratoderma.American Journal of Human Genetics. 2013; 93: 330-335Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), AQP5 was localized to the membrane of suprabasal keratinocytes in normal and diffuse NEPPK palm, whilst it displayed a diffuse, cytoplasmic localization in body skin. CD44 expression was observed in the plasma membrane of both basal and suprabasal keratinocytes in normal and diffuse NEPPK palm. In a similar fashion to AQP5, CD44 also displayed a more diffuse expression in body skin, suggestive of a specific role of these proteins in palmoplantar epidermis. It is worth noting that the expression levels of AQP5 and CD44 in AQP5Ala38Glu diffuse NEPPK palm were comparably lower to those in AQP5Ile177Phe and normal palm (Figure 2D,E). In contrast, both AQP5 and CD44 protein levels were increased in AQP5Ile177Phe palm compared to normal palm. Given the association between CD44 and actin organization, we hypothesize that elevated levels of CD44 are likely having an impact in increased actin stress fibre formation in AQP5Ile177Phe palm skin. PLA was used to determine whether AQP5 and CD44 interact in the epidermis (Figure 2F). PLA signals were observed in the membrane of suprabasal keratinocytes in both normal and diffuse NEPPK palm. Although a few PLA signals were observed in body skin, they mostly appeared diffuse and cytoplasmic, suggesting that AQP5-CD44 interactions have a site-specific role in the palmoplantar epidermis. In addition, the PLA signal intensity in AQP5Ile177Phe diffuse NEPPK palm was noticeably higher than in normal palm (Figure 2H), indicative of increased interactions between AQP5Ile177Phe variant protein and CD44. As hyaluronic acid (HA) is a major binding partner of CD44, staining of HA in body and palm skin using biotinylated hyaluronan binding protein (HABP) was performed (Figure 2G). Confocal microscopy revealed a substantial increase in HA levels in AQP5Ile177Phe palm compared to normal palm and body skin. No apparent difference in HA levels was observed between AQP5Ala38Glu palm and controls (Figure 2I). HA/CD44 interaction has been associated with RhoA/ROCK activation, resulting in actin polymerization and filament bundling (Zhang et al., 2021Zhang H. Cai W. Shao X. Regulation of aquaporin-3 water permeability by hyaluronan.Physical chemistry Chemical Physics. 2021; 23: 25706-25711Crossref PubMed Google Scholar). RhoA is also an upstream regulator of pMLC2, which in turn is involved in induction of contractile actin stress fibre assembly. Increased contractility in differentiated keratinocytes in the skin is reported to induce stem cell hyperproliferation, resulting in increased epidermal thickness (Ning et al., 2021Ning W. Muroyama A. Li H. Lechler T. Differentiated Daughter Cells Regulate Stem Cell Proliferation and Fate through Intra-tissue Tension.Cell stem cell. 2021; 28: 436-452Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). We therefore hypothesize that increased levels of HA in AQP5Ile177Phe diffuse NEPPK skin could account for the observed increased actin stress fibres and hyperproliferative phenotype. Here, we present evidence that some diffuse NEPPK-associated AQP5 mutations result in a dysregulated actin cytoskeleton, characterized by increased actin stress fibres. This may be via an increased interaction between variant AQP5 proteins and CD44, a known regulator of actin remodelling. In contrast, AQP5Ala38Glu palm displayed reduced levels of AQP5, CD44 and PLA signal intensity compared to normal and AQP5Ile177Phe palm. Phenotypically, AQP5Ala38Glu variant palmoplantar skin has a comparably less thickened epidermis. In addition, the Ala38Glu variant is located in the extracellular surface of AQP5, whilst Ile177Phe is found near the ar/R constriction point inside the channel (Blaydon et al., 2013Blaydon D.C. Lind L.K. Plagnol V. Linton K.J. Smith F.J.D. Wilson N.J. et al.Mutations in AQP5, encoding a water channel protein, cause autosomal-dominant diffuse nonepidermolytic palmoplantar keratoderma.American Journal of Human Genetics. 2013; 93: 330-335Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Therefore, we propose that these mutations may have different impacts on AQP5 conformation, resulting in altered interactions with CD44 and differential downstream impacts on hyperkeratosis. For instance, the replacement of isoleucine by a bulkier phenylalanine in the AQP5Ile177Phe mutation may cause an effect in the size of the pore via steric hindrance. On the contrary, the replacement of alanine 38 by a charged glutamine in the extracellular loop of AQP5Ala38Glu is hypothesized to have an impact on the stability of this water channel (Blaydon et al., 2013Blaydon D.C. Lind L.K. Plagnol V. Linton K.J. Smith F.J.D. Wilson N.J. et al.Mutations in AQP5, encoding a water channel protein, cause autosomal-dominant diffuse nonepidermolytic palmoplantar keratoderma.American Journal of Human Genetics. 2013; 93: 330-335Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Due to the rareness of this condition and limited access to patient-derived material, we were unable to explore these findings in skin samples harbouring the AQP5Ile45Ser variant. Interestingly, molecular dynamic simulations have reported a role of HA in the regulation of water permeability of AQP3, another member of the Aquaporin protein family expressed in skin (Zhang et al., 2021Zhang H. Cai W. Shao X. Regulation of aquaporin-3 water permeability by hyaluronan.Physical chemistry Chemical Physics. 2021; 23: 25706-25711Crossref PubMed Google Scholar). Altogether, this data proposes a role for AQP5 in the regulation of actin cytoskeleton dynamics in the skin, whilst deepening our understanding of the unique molecular characteristics of the palmoplantar epidermis and how it contrasts to body skin. No large datasets were generated or analyzed during this study. EOT is a consultant for Kamari Pharma, Palvella Therapeutics and Unilever (unrelated to this work) and receives grants to her institution from Kamari Pharma, Palvella Therapeutics and Azitra. The remaining authors state no conflict of interest. This work is supported by a grant awarded to D.C.B. from the British Skin Foundation (011/S/18) as well as by a grant awarded to D.P.K. from the Medical Research Council (MR/S009914/1). The authors would like to thank Dr Anita Lundström (Dermatology and Venereology, Department of Public Health and Clinical Medicine, Umeå University, Umeå, Sweden) for providing the AQP5Ala38Glu palm biopsies. The authors would also like to thank Dr Roberto Buccafusca from the School of Biological and Behavioural Chemical Sciences (SBBS) Mass Spectrometry Laboratory and the Blizard Advanced Light Microscopy core facilities for their collaboration in this project. Author contribution statement (CRediT-compliant) Conceptualization: DCB, DPK; Investigation: LRdC, DCB; Funding acquisition: DCB, DPK; Supervision: DCB, DPK; Resources: APS, EAO; Writing – original draft: LRdC; Writing – review & editing: APS, EAO, DPK, DCB. Download .pdf (.24 MB) Help with pdf files