Discordant growth of nasal cartilage and bone contributes to phenotypic variability of the skull in a mouse model.

BACKGROUND: We previously determined a nonlinear relationship between connexin 43 (Cx43) function and craniofacial phenotypic variation in the mutant mouse model G60S/+, and that this variation was driven by nasal bone deviation. While nonlinearities in the genotype-phenotype map appear to be common, few studies have looked at the developmental processes that underlie this nonlinearity. Here, we investigated the potential tissue-level developmental determinants of the variation in nasal bone phenotype in G60S/+ mice through postnatal development. RESULTS: The deviated nasal bone phenotype arises by postnatal day 21 and becomes more severe by 3 months in G60S/+ mice. Measures of nasal bone remodeling including the number of osteoclasts, mineralizing surface, mineral apposition rate, and bone formation rate are significantly greater in G60S/+ mice compared to wild-type mice at 2 months, but these differences do not correspond with nasal bone deviation. The degree of nasal bone deviation does significantly and negatively correlate with the ratio between nasal bone and cartilaginous nasal septum length. CONCLUSIONS: Our findings indicate that the mean phenotypic changes observed between G60S/+ and wild-type mice are due to reduced bone growth, but the increased phenotypic variation found within mutant mice is due to discordant growth between nasal cartilage and bone.

Pannexin 3 deletion reduces fat accumulation and inflammation in a sex-specific manner.

BACKGROUND: Pannexin 3 (PANX3) is a channel-forming glycoprotein that enables nutrient-induced inflammation in vitro, and genetic linkage data suggest that it regulates body mass index. Here, we characterized inflammatory and metabolic parameters in global Panx3 knockout (KO) mice in the context of forced treadmill running (FEX) and high-fat diet (HFD). METHODS: C57BL/6N (WT) and KO mice were randomized to either a FEX running protocol or no running (SED) from 24 until 30 weeks of age. Body weight was measured biweekly, and body composition was measured at 24 and 30 weeks of age. Male WT and KO mice were fed a HFD from 12 to 28 weeks of age. Metabolic organs were analyzed for a panel of inflammatory markers and PANX3 expression. RESULTS: In females there were no significant differences in body composition between genotypes, which could be due to the lack of PANX3 expression in female white adipose tissue, while male KOs fed a chow diet had lower body weight and lower fat mass at 24 and 30 weeks of age, which was reduced to the same extent as 6 weeks of FEX in WT mice. In addition, male KO mice exhibited significantly lower expression of multiple pro-inflammatory genes in white adipose tissue compared to WT mice. While on a HFD body weight differences were insignificant, multiple inflammatory genes were significantly different in quadriceps muscle and white adipose tissue resulting in a more anti-inflammatory phenotype in KO mice compared to WT. The lower fat mass in male KO mice may be due to significantly fewer adipocytes in their subcutaneous fat compared to WT mice. Mechanistically, adipose stromal cells (ASCs) cultured from KO mice grow significantly slower than WT ASCs. CONCLUSION: PANX3 is expressed in male adult mouse adipose tissue and may regulate adipocyte numbers, influencing fat accumulation and inflammation.

Interrogation of Carboxy-Terminus Localized GJA1 Variants Associated with Erythrokeratodermia Variabilis et Progressiva.

Although inherited GJA1 (encoding Cx43) gene mutations most often lead to oculodentodigital dysplasia and related disorders, four variants have been linked to erythrokeratodermia variabilis et progressiva (EKVP), a skin disorder characterized by erythematous and hyperkeratotic lesions. While two autosomal-dominant EKVP-linked GJA1 mutations have been shown to lead to augmented hemichannels, the consequence(s) of keratinocytes harboring a de novo P283L variant alone or in combination with a de novo T290N variant remain unknown. Interestingly, these variants reside within or adjacent to a carboxy terminus polypeptide motif that has been shown to be important in regulating the internalization and degradation of Cx43. Cx43-rich rat epidermal keratinocytes (REKs) or Cx43-ablated REKs engineered to express fluorescent protein-tagged P283L and/or T290N variants formed prototypical gap junctions at cell-cell interfaces similar to wildtype Cx43. Dye coupling and dye uptake studies further revealed that each variant or a combination of both variants formed functional gap junction channels, with no evidence of augmented hemichannel function or induction of cell death. Tracking the fate of EKVP-associated variants in the presence of the protein secretion blocker brefeldin A, or an inhibitor of protein synthesis cycloheximide, revealed that P283L or the combination of P283L and T290N variants either significantly extended Cx43 residency on the cell surface of keratinocytes or delayed its degradation. However, caution is needed in concluding that this modest change in the Cx43 life cycle is sufficient to cause EKVP, or whether an additional underlying mechanism or another unidentified gene mutation is contributing to the pathogenesis found in patients. This question will be resolved if further patients are identified where whole exome sequencing reveals a Cx43 P283L variant alone or, in combination with a T290N variant, co-segregates with EKVP across several family generations.

Connexin 43 contributes to phenotypic robustness of the mouse skull.

BACKGROUND: We compared skull shape and variation among genetically modified mice that exhibit different levels of connexin43 (Cx43) channel function, to determine whether Cx43 contributes to craniofacial phenotypic robustness. Specifically, we used two heterozygous mutant mouse models (G60S/+ and I130T/+) that, when compared to their wildtype counterparts, have an ~80% and ~50% reduction in Cx43 function, respectively. RESULTS: Both mutant strains showed significant differences in skull shape compared to wildtype littermates and while these differences were more severe in the G60S/+ mouse, shape differences were localized to similar regions of the skull in both mutants. However, increased skull shape variation was observed in G60S/+ mutants only. Additionally, covariation of skull structures was disrupted in the G60S/+ mutants only, indicating that while a 50% reduction in Cx43 function is sufficient to cause a shift in mean skull shape, the threshold for Cx43 function for disrupting craniofacial phenotypic robustness is lower. CONCLUSIONS: Collectively, our results indicate Cx43 can contribute to phenotypic robustness of the skull through a nonlinear relationship between Cx43 gap junctional function and phenotypic outcomes.

Effects of advanced maternal age and acute prenatal alcohol exposure on mouse offspring growth and craniofacial phenotype.

BACKGROUND: Prenatal alcohol exposure (PAE) can result in developmental defects that include growth restriction, craniofacial anomalies, and cognitive behavioral deficits, though the presence and severity of these adverse outcomes can vary dramatically among exposed individuals. Preclinical animal models have demonstrated that the dose and timing of PAE account for much, but not all, of this phenotypic variation, suggesting that additional factors mitigate the effects of PAE. Here, we used a mouse model to investigate whether maternal age modulates the effects of PAE on the severity and variation in offspring growth and craniofacial outcomes. METHODS: Nulliparous C57BL/6N dams received either an intraperitoneal injection of ethanol (EtOH) or vehicle solution on gestational day 7.5. Dams were divided into four groups: (1) EtOH-treated young dams (6 to 10 weeks); (2) control young dams; (3) EtOH-treated old dams (6 to 7 months); and (4) old control dams. Neonate offspring growth restriction was measured through body mass and organ-to-body mass ratios, while skeletal craniofacial features were imaged using micro-CT and analyzed for size, shape, and variation. RESULTS: PAE and advanced maternal age each increased the risk of low birthweight and growth restriction in offspring, but these factors in combination changed the nature of the growth restriction. Similarly, both PAE and advanced maternal age individually caused changes to craniofacial morphology such as smaller skull size, dysmorphic skull shape, and greater skull shape variation and asymmetry. Interestingly, while the combination of PAE and advanced maternal age did not affect mean skull shape or size, it significantly increased the variation and asymmetry of those measures. CONCLUSION: Our results indicate that maternal age modulates the effects of PAE, but that the effects of this combination on offspring outcomes are more complex than simply scaling the effects of either factor.

Mice harbouring an oculodentodigital dysplasia-linked Cx43 G60S mutation have severe hearing loss.

Given the importance of connexin43 (Cx43, encoded by GJA1) function in the central nervous system and sensory organ processing, we proposed that it would also be crucial in auditory function. To that end, hearing was examined in two mouse models of oculodentodigital dysplasia that globally express GJA1 mutations resulting in mild or severe loss of Cx43 function. Although Cx43I130T/+ mutant mice, with ∼50% Cx43 channel function, did not have any hearing loss, Cx43G60S/+ mutant mice, with ∼20% Cx43 channel function, had severe hearing loss. There was no evidence of inner ear sensory hair cell loss, suggesting that the mechanism for Cx43-linked hearing loss lies downstream in the auditory pathway. Since evidence suggests that Cx26 function is essential for hearing and may be protective against noise-induced hearing loss, we challenged Cx43I130T/+ mice with a loud noise and found that they had a similar susceptibility to noise-induced hearing loss to that found in controls, suggesting that decreased Cx43 function does not sensitize the mice for environmentally induced hearing loss. Taken together, this study suggests that Cx43 plays an important role in baseline hearing and is essential for auditory processing.This article has an associated First Person interview with the first author of the paper.

Effects of Reduced Connexin43 Function on Mandibular Morphology and Osteogenesis in Mutant Mouse Models of Oculodentodigital Dysplasia.

Mutations in the gene encoding the gap-junctional protein connexin43 (Cx43) are the cause of the human disease oculodentodigital dysplasia (ODDD). The mandible is often affected in this disease, with clinical reports describing both mandibular overgrowth and conversely, retrognathia. These seemingly opposing observations underscore our relative lack of understanding of how ODDD affects mandibular morphology. Using two mutant mouse models that mimic the ODDD phenotype (I130T/+ and G60S/+), we sought to uncover how altered Cx43 function may affect mandibular development. Specifically, mandibles of newborn mice were imaged using micro-CT, to enable statistical comparisons of shape. Tissue-level comparisons of key regions of the mandible were conducted using histomorphology, and we quantified the mRNA expression of several cartilage and bone cell differentiation markers. Both G60S/+ and I130T/+ mutant mice had altered mandibular morphology compared to their wildtype counterparts, and the morphological effects were similarly localized for both mutants. Specifically, the biggest phenotypic differences in mutant mice were focused in regions exposed to mechanical forces, such as alveolar bone, muscular attachment sites, and articular surfaces. Histological analyses revealed differences in ossification of the intramembranous bone of the mandibles of both mutant mice compared to their wildtype littermates. However, chondrocyte organization within the secondary cartilages of the mandible was unaffected in the mutant mice. Overall, our results suggest that the morphological differences seen in G60S/+ and I130T/+ mouse mandibles are due to delayed ossification and suggest that mechanical forces may exacerbate the effects of ODDD on the skeleton.

Differential effects of pannexins on noise-induced hearing loss.

Hearing loss, including noise-induced hearing loss, is highly prevalent and severely hinders an individual's quality of life, yet many of the mechanisms that cause hearing loss are unknown. The pannexin (Panx) channel proteins, Panx1 and Panx3, are regionally expressed in many cell types along the auditory pathway, and mice lacking Panx1 in specific cells of the inner ear exhibit hearing loss, suggesting a vital role for Panxs in hearing. We proposed that Panx1 and/or Panx3 null mice would exhibit severe hearing loss and increased susceptibility to noise-induced hearing loss. Using the auditory brainstem response, we surprisingly found that Panx1-/- and Panx3-/- mice did not harbor hearing or cochlear nerve deficits. Furthermore, while Panx1-/- mice displayed no protection against loud noise-induced hearing loss, Panx3-/- mice exhibited enhanced 16- and 24-kHz hearing recovery 7 days after a loud noise exposure (NE; 12 kHz tone, 115 dB sound pressure level, 1 h). Interestingly, Cx26, Cx30, Cx43, and Panx2 were up-regulated in Panx3-/- mice compared with wild-type and/or Panx1-/- mice, and assessment of the auditory tract revealed morphological changes in the middle ear bones of Panx3-/- mice. It is unclear if these changes alone are sufficient to provide protection against loud noise-induced hearing loss. Contrary to what we expected, these data suggest that Panx1 and Panx3 are not essential for baseline hearing in mice tested, but the therapeutic targeting of Panx3 may prove protective against mid-high-frequency hearing loss caused by loud NE.

Mechanisms of NDV-3 vaccine efficacy in MRSA skin versus invasive infection.

Increasing rates of life-threatening infections and decreasing susceptibility to antibiotics urge development of an effective vaccine targeting Staphylococcus aureus. This study evaluated the efficacy and immunologic mechanisms of a vaccine containing a recombinant glycoprotein antigen (NDV-3) in mouse skin and skin structure infection (SSSI) due to methicillin-resistant S. aureus (MRSA). Compared with adjuvant alone, NDV-3 reduced abscess progression, severity, and MRSA density in skin, as well as hematogenous dissemination to kidney. NDV-3 induced increases in CD3+ T-cell and neutrophil infiltration and IL-17A, IL-22, and host defense peptide expression in local settings of SSSI abscesses. Vaccine induction of IL-22 was necessary for protective mitigation of cutaneous infection. By comparison, protection against hematogenous dissemination required the induction of IL-17A and IL-22 by NDV-3. These findings demonstrate that NDV-3 protective efficacy against MRSA in SSSI involves a robust and complementary response integrating innate and adaptive immune mechanisms. These results support further evaluation of the NDV-3 vaccine to address disease due to S. aureus in humans.

Global deletion of Panx3 produces multiple phenotypic effects in mouse humeri and femora.

Pannexins form single-membrane channels that allow passage of small molecules between the intracellular and extracellular compartments. Of the three pannexin family members, Pannexin3 (Panx3) is the least studied but is highly expressed in skeletal tissues and is thought to play a role in the regulation of chondrocyte and osteoblast proliferation and differentiation. The purpose of our study is to closely examine the in vivo effects of Panx3 ablation on long bone morphology using micro-computed tomography. Using Panx3 knockout (KO) and wildtype (WT) adult mice, we measured and compared aspects of phenotypic shape, bone mineral density (BMD), cross-sectional geometric properties of right femora and humeri, and lean mass. We found that KO mice have absolutely and relatively shorter diaphyseal shafts compared with WT mice, and relatively larger areas of muscle attachment sites. No differences in BMD or lean mass were found between WT and KO mice. Interestingly, KO mice had more robust femora and humeri compared with WT mice when assessed in cross-section at the midshaft. Our results clearly show that Panx3 ablation produces phenotypic effects in mouse femora and humeri, and support the premise that Panx3 has a role in regulating long bone growth and development.

Nonredundant Roles of Interleukin-17A (IL-17A) and IL-22 in Murine Host Defense against Cutaneous and Hematogenous Infection Due to Methicillin-Resistant Staphylococcus aureus.

Staphylococcus aureus is the leading cause of skin and skin structure infections (SSSI) in humans. Moreover, the high frequency of recurring SSSI due to S. aureus, particularly methicillin-resistant S. aureus (MRSA) strains, suggests that infection induces suboptimal anamnestic defenses. The present study addresses the hypothesis that interleukin-17A (IL-17A) and IL-22 play distinct roles in immunity to cutaneous and invasive MRSA infection in a mouse model of SSSI. Mice were treated with specific neutralizing antibodies against IL-17A and/or IL-22 and infected with MRSA, after which the severity of infection and host immune response were determined. Neutralization of either IL-17A or IL-22 reduced T cell and neutrophil infiltration and host defense peptide elaboration in lesions. These events corresponded with increased abscess severity, MRSA viability, and CFU density in skin. Interestingly, combined inhibition of IL-17A and IL-22 did not worsen abscesses but did increase gamma interferon (IFN-γ) expression at these sites. The inhibition of IL-22 led to a reduction in IL-17A expression, but not vice versa. These results suggest that the expression of IL-17A is at least partially dependent on IL-22 in this model. Inhibition of IL-17A but not IL-22 led to hematogenous dissemination to kidneys, which correlated with decreased T cell infiltration in renal tissue. Collectively, these findings indicate that IL-17A and IL-22 have complementary but nonredundant roles in host defense against cutaneous versus hematogenous infection. These insights may support targeted immune enhancement or other novel approaches to address the challenge of MRSA infection.

Myogenic bladder defects in mouse models of human oculodentodigital dysplasia.

To date, over 65 mutations in the gene encoding Cx43 (connexin43) have been linked to the autosomal-dominant disease ODDD (oculodentodigital dysplasia). A subset of these patients experience bladder incontinence which could be due to underlying neurogenic deterioration or aberrant myogenic regulation. BSMCs (bladder smooth muscle cells) from wild-type and two Cx43 mutant lines (Cx43(G60S) and Cx43(I130T)) that mimic ODDD exhibit a significant reduction in total Cx43. Dye transfer studies revealed that the G60S mutant was a potent dominant-negative inhibitor of co-expressed Cx43, a property not equally shared by the I130T mutant. BSMCs from both mutant mouse strains were defective in their ability to contract, which is indicative of phenotype changes due to harbouring the Cx43 mutants. Upon stretching, Cx43 levels were significantly elevated in controls and mutants containing BSMCs, but the non-muscle myosin heavy chain A levels were only reduced in cells from control mice. Although the Cx43(G60S) mutant mice showed no difference in voided urine volume or frequency, the Cx43(I130T) mice voided less frequently. Thus, similar to the diversity of morbidities seen in ODDD patients, genetically modified mice also display mutation-specific changes in bladder function. Furthermore, although mutant mice have compromised smooth muscle contraction and response to stretch, overriding bladder defects in Cx43(I130T) mice are likely to be complemented by neurogenic changes.

The severity of mammary gland developmental defects is linked to the overall functional status of Cx43 as revealed by genetically modified mice.

Genetically modified mice mimicking ODDD (oculodentodigital dysplasia), a disease characterized by reduced Cx43 (connexin 43)-mediated gap junctional intercellular communication, represent an in vivo model to assess the role of Cx43 in mammary gland development and function. We previously reported that severely compromised Cx43 function delayed mammary gland development and impaired milk ejection in mice that harboured a G60S Cx43 mutant, yet there are no reports of lactation defects in ODDD patients. To address this further, we obtained a second mouse model of ODDD expressing an I130T Cx43 mutant to assess whether a mutant with partial gap junction channel activity would be sufficient to retain mammary gland development and function. The results of the present study show that virgin Cx43I130T/+ mice exhibited a temporary delay in ductal elongation at 4 weeks. In addition, Cx43I130T/+ mice develop smaller mammary glands at parturition due to reduced cell proliferation despite similar overall gland architecture. Distinct from Cx43G60S/+ mice, Cx43I130T/+ mice adequately produce and deliver milk to pups, suggesting that milk ejection is unaffected. Thus the present study suggests that a loss-of-function mutant of Cx43 with partial gap junction channel coupling conductance results in a less severe mammary gland phenotype, which may partially explain the lack of reported lactation defects associated with ODDD patients.

Analysis of Proliferation, Apoptosis, and Motility in Mouse Embryonic Melanocytic Precursor Cells.

In this chapter, we provide a method to purify and culture embryonic melanocytic stem cells that express green fluorescent protein in a cell-type specific manner. Isolation of melanocytic lineage cell populations that are >98% pure is accomplished through the use of GFP-based fluorescence activated cell sorting. We also provide a method to culture the purified melanoblasts and to analyze their proliferation, apoptosis, and motility properties.

Global pannexin 1 deletion increases tumor-infiltrating lymphocytes in the BRAF/Pten mouse melanoma model.

Immunotherapies for malignant melanoma seek to boost the anti-tumoral response of CD8+ T cells, but have a limited patient response rate, in part due to limited tumoral immune cell infiltration. Genetic or pharmacological inhibition of the pannexin 1 (PANX1) channel-forming protein is known to decrease melanoma cell tumorigenic properties in vitro and ex vivo. Here, we crossed Panx1 knockout (Panx1-/-) mice with the inducible melanoma model BrafCA, PtenloxP, Tyr::CreERT2 (BPC). We found that deleting the Panx1 gene in mice does not reduce BRAF(V600E)/Pten-driven primary tumor formation or improve survival. However, tumors in BPC-Panx1-/- mice exhibited a significant increase in the infiltration of CD8+ T lymphocytes, with no changes in the expression of early T-cell activation marker CD69, lymphocyte activation gene 3 protein (LAG-3) checkpoint receptor, or programmed cell death ligand-1 (PD-L1) in tumors when compared to the BPC-Panx1+/+ genotype. Our results suggest that, although Panx1 deletion does not overturn the aggressive BRAF/Pten-driven melanoma progression in vivo, it does increase the infiltration of effector immune T-cell populations in the tumor microenvironment. We propose that PANX1-targeted therapy could be explored as a strategy to increase tumor-infiltrating lymphocytes to boost anti-tumor immunity.

Novel Pannexin 1 isoform is increased in cancer.

Pannexin 1 (PANX1) is upregulated in many cancers, where its activity and signalling promote tumorigenic properties. Here, we report a novel ∼25 kDa isoform of human PANX1 (hPANX1-25K) which lacks the N-terminus and was detected in several human cancer cell lines including melanoma, osteosarcoma, breast cancer and glioblastoma multiforme. This isoform was increased upon hPANX1 CRISPR/Cas9 deletion targeting the first exon near M1, suggesting a potential alternative translation initiation (ATI) site. hPANX1-25K was confirmed to be a hPANX1 isoform via mass spectrometry, can be N-linked glycosylated at N254, and can interact with both ß-catenin and full length hPANX1. A double deletion of hPANX1 and hPANX1-25K reduces cell growth and viability in cancer cells. hPANX1-25K is prevalent throughout melanoma progression, and its levels are increased in squamous cell carcinoma cells and patient-derived tumours, compared to keratinocytes and normal skin, indicating that it may be differentially regulated in normal and cancer cells.

Decidual angiogenesis and placental orientation are altered in mice heterozygous for a dominant loss-of-function Gja1 (connexin43) mutation.

Connexin43 (CX43), encoded by Gja1 in the mouse, is highly expressed in decidual cells and is known to be important for the transformation of stromal cells into the compact decidua and for neoangiogenesis. Here we investigated if the dominant Gja1(Jrt) mutation encoding CX43(G60S) in mice, which results in a phenotype resembling oculodentodigital dysplasia in humans, has an impact on decidualization, angiogenesis, and implantation. We found a reduced mean weight of fetuses at Gestational Day 17.5 in dams carrying this mutation, with the growth deficiency being independent of fetal genotype. Although the mutant implantation sites exhibited a reduction in CX43 protein, with most immunoreactivity being cytoplasmic, the decidua was morphologically intact at Embryonic Days 5.5 to 7.5. However, the mutation resulted in enhanced and irregular angiogenesis and an increased level of expression of the angiogenic factor-encoding genes Vegfa, Flt1, Kdr, and Fgf2 as well as the prolactin-related gene Prl6a. Moreover, immunolocalization of VEGFA, FLT1, and KDR revealed a homogeneous distribution pattern in the mesometrial as well as antimesometrial decidua of the mutants. Most obviously, uterine NK cells are drastically diminished in the mesometrial decidua of the mutant mice. Invasion of ectoplacental cone cells was disoriented, and placentation was established more laterally in the implantation chambers. It was concluded that the CX43(G60S) mutant impairs control of decidual angiogenesis, leading to dysmorphic placentation and fetal growth restriction. This phenomenon could contribute to the reduced fetal weights and viability of pups born of Gja1(Jrt)/+ dams.

Connexins and steroidogenesis in mouse Leydig cells.

Connexin43 has been recognized as forming gap junctions in Leydig cells. However, previous work has shown that mouse Leydig cells lacking this connexin do not suffer any limitation of their ability to produce testosterone when stimulated with luteinizing hormone. The objective of this study was to identify additional connexins in mouse Leydig cells that could be required for steroidogenesis. A reverse transcription - polymerase chain reaction screen involving isolated adult Leydig cells identified connexin36 and connexin45 as expressed along with connexin43. Treatment of dissociated testes with carbenoxolone, a nonspecific blocker of gap junctional coupling, significantly reduced testosterone output as did treatment with quinine, which disrupts coupling provided by connexin36 and connexin45 gap junctions but not those composed of connexin43, indicating that either or both of connexins 36 and 45 could be involved in supporting Leydig cell steroidogenesis. Immunolabeling of adult mouse testis sections confirmed the localization of connexin36 along with connexin43 in Leydig cell gap junctions but not connexin45, which is distributed throughout the cells. It was concluded that connexin36, connexin43, and connexin45 are coexpressed in Leydig cells with connexins 36 and 43 contributing to gap junctions. The role of connexin45 remains to be elucidated.

Isolation, Purification, and Culture of Embryonic Melanoblasts from Green Fluorescent Protein-expressing Reporter Mice.

In this article, we provide a method to isolate embryonic melanoblasts from reporter mouse strains. The mice from which these cells are isolated are bred into the ROSA26mT/mG reporter background, which results in green fluorescent protein (GFP) expression in the targeted melanoblast population. These cells are isolated and purified by fluorescence-activated cell sorting using GFP fluorescence. We also provide a method to culture the purified melanoblasts for further analysis. This method yields > 99% purity melanoblasts specifically targeted, and can be used for a variety of studies, including gene expression, clonogenic experiments, and biological assays, such as viability, capacity for directional migration, or differentiation into melanin-producing melanocytic cells.

PANX3 Channels Regulate Architecture, Adhesion, Barrier Function, and Inflammation in the Skin.

The channel-forming glycoprotein PANX3 functions in cutaneous wound healing and keratinocyte differentiation, but its role in maintaining skin homeostasis through aging is not yet understood. We found that PANX3 is absent in newborn skin but becomes upregulated with age. We characterized the skin of global Panx3-knockout (KO) mice and found that KO dorsal skin showed sex differences at different ages but generally had reduced dermal and hypodermal areas compared with age-matched controls. Transcriptomic analysis of the KO epidermis revealed reduced E-cadherin stabilization and Wnt signaling compared with that of wild-type, consistent with the inability of primary KO keratinocytes to adhere in culture and diminished epidermal barrier function in KO mice. We also observed increased inflammatory signaling in the KO epidermis and a higher incidence of dermatitis in aged KO mice compared with that in wild-type controls. These findings suggest that during skin aging, PANX3 is critical in the maintenance of dorsal skin architecture, keratinocyte cell-cell and cell-matrix adhesion, and inflammatory skin responses.