Fluid shear stress stimulates ATP release without regulating purinergic gene expression in the renal inner medullary collecting duct.

In the kidney, the flow rate of the pro-urine through the renal tubules is highly variable. The tubular epithelial cells sense these variations in pro-urinary flow rate in order to regulate various physiological processes, including electrolyte reabsorption. One of the mechanosensitive pathways activated by flow is the release of ATP, which can then act as a autocrine or paracrine factor. Increased ATP release is observed in various kidney diseases, among others autosomal dominant polycystic kidney disease (ADPKD). However, the mechanisms underlying flow-induced ATP release in the collecting duct, especially in the inner medullary collecting duct, remain understudied. Using inner medullary collecting duct 3 (IMCD3) cells in a microfluidic setup, we show here that administration of a high flow rate for 1 min results in an increased ATP release compared to a lower flow rate. Although the ATP release channel pannexin-1 contributed to flow-induced ATP release in Pkd1-/- IMCD3 cells, it did not in wildtype IMCD3 cells. In addition, flow application increased the expression of the putative ATP release channel connexin-30.3 (CX30.3) in wildtype and Pkd1-/- IMCD3 cells. However, CX30.3 knockout IMCD3 cells exhibited a similar flow-induced ATP release as wildtype IMCD3 cells, suggesting that CX30.3 does not drive flow-induced ATP release in wildtype IMDC3 cells. Collectively, our results show differential mechanisms underlying flow-induced ATP release in wildtype and Pkd1-/- IMCD3 cells and further strengthen the link between ADPKD and pannexin-1-dependent ATP release.

Erythrokeratodermia variabilis et progressiva due to a novel mutation in GJB4.

Erythrokeratodermia variabilis et progressiva (EKVP) is a rare genodermatosis of clinical and genetic heterogeneity, characterized by the manifestations of localized or disseminated persistent hyperkeratotic plagues and stationary to migratory transient erythematous patches. The majority of EKVP cases display an autosomal dominant mode of inheritance with incomplete penetrance, although recessive transmission has also been described. Mutations associated with EKVP have been primarily detected in connexin (Cx) genes. We herein reported a Chinese sporadic case of late-onset EKVP with a novel heterozygous missense mutation c.109G>A (p.V37M) in GJB4 (Cx30.3) gene, which resulted in a significant reduction of GJB4 expression in the epidermis of the patient. In accordance, while wild-type GJB4 localized at the cell membrane of HeLa cells forming intercellular junctions and intracellular puncta, V37M mutant variant was diffusely expressed within HeLa cells at a considerably lower level. Our findings reveal an essential role of GJB4 in the pathogenesis of EKVP and provides insights into the therapeutic potential of the disease.

Diverse pattern of gap junction beta-2 and gap junction beta-4 genes mutations and lack of contribution of DFNB21, DFNB24, DFNB29, and DFNB42 loci in autosomal recessive nonsyndromic hearing loss patients in Hormozgan, Iran.

BACKGROUND: We aimed to determine the contribution of four DFNB loci and mutation analysis of gap junction beta-2 (GJB2) and GJB4 genes in autosomal recessive nonsyndromic hearing loss (ARNSHL) in South of Iran. MATERIALS AND METHODS: A total of 36 large ARNSHL pedigrees with at least two affected subjects were enrolled in the current study. The GJB2 and GJB4 genes mutations were screened using direct sequencing method. The GJB2 and GJB4 negative families were analyzed for the linkage to DFNB21, DFNB24, DFNB29, and DFNB42 loci by genotyping the corresponding STR markers using polymerase chain reaction-PAGE method. RESULTS: We found a homozygous nonsense mutation W77X and a homozygous missense mutation C169W in 5.55% of studied families in GJB2 and GJB4 genes, respectively. Five heterozygous mutations including V63G, A78T, and R127H in GJB2 gene, and R103C and R227W in GJB4 gene were detected. We identified two novel variations V63G in GJB2 and R227W in GJB4. In silico analysis predicted that both novel variations are deleterious mutations. We did not unveil any linkage between DFNB21, DFNB24, DFNB29, and DFNB42 loci and ARNSHL among studied families. CONCLUSION: This is the first report of GJB2 and GJB4 mutations from Hormozgan population. According to the previous publications regarding GJB2 and GJB4 mutations, the distribution of the mutations is different from other parts of Iran that should be considered in primary health-care programs. Further investigations are needed to evaluate the contribution of other loci in ARNSHL subjects in South of Iran.

GJB4 variants linked to skin disease exhibit a trafficking deficiency en route to gap junction formation that can be restored by co-expression of select connexins.

Epidermal keratinocytes are enriched with at least nine connexins that are key regulators of epidermal homeostasis. The role of Cx30.3 in keratinocytes and epidermal health became evident when fourteen autosomal dominant mutations in the Cx30.3-encoding GJB4 gene were linked to a rare and incurable skin disorder called erythrokeratodermia variabilis et progressiva (EKVP). While these variants are linked to EKVP, they remain largely uncharacterized hindering therapeutic options. In this study, we characterize the expression and functional status of three EKVP-linked Cx30.3 mutants (G12D, T85P, and F189Y) in tissue-relevant and differentiation-competent rat epidermal keratinocytes. We found that GFP-tagged Cx30.3 mutants were non-functional likely due to their impaired trafficking and primary entrapment within the endoplasmic reticulum (ER). However, all mutants failed to increase BiP/GRP78 levels suggesting they were not inducing an unfolded protein response. FLAG-tagged Cx30.3 mutants were also trafficking impaired yet occasionally exhibited some capacity to assemble into gap junctions. The pathological impact of these mutants may extend beyond their trafficking deficiencies as keratinocytes expressing FLAG-tagged Cx30.3 mutants exhibited increased propidium iodide uptake in the absence of divalent cations. Attempts to rescue the delivery of trafficking impaired GFP-tagged Cx30.3 mutants into gap junctions by chemical chaperone treatment were ineffective. However, co-expression of wild type Cx30.3 greatly enhanced the assembly of Cx30.3 mutants into gap junctions, although endogenous levels of Cx30.3 do not appear to prevent the skin pathology found in patients harboring these autosomal dominant mutations. In addition, a spectrum of connexin isoforms (Cx26, Cx30, and Cx43) exhibited the differential ability to trans-dominantly rescue the assembly of GFP-tagged Cx30.3 mutants into gap junctions suggesting a broad range of connexins found in keratinocytes may favourably interact with Cx30.3 mutants. We conclude that selective upregulation of compatible wild type connexins in keratinocytes may have potential therapeutic value in rescuing epidermal defects invoked by Cx30.3 EKVP-linked mutants.

The Functional Role of CONNEXIN 26 Mutation in Nonsyndromic Hearing Loss, Demonstrated by Zebrafish Connexin 30.3 Homologue Model.

Nonsyndromic hearing loss (NSHL) is of great clinical importance, and mutations in the GJB2 gene and the encoded human CONNEXIN 26 (CX26) protein play important roles in the genetic pathogenesis. The CX26 p.R184Q mutation was shown to be a dominant-negative effect in our previous study. Previously, we also demonstrated that zebrafish Cx30.3 is orthologous to human CX26. In the present study, we established transgenic zebrafish models with mutated Cx30.3 specifically expressed in the supporting cells of zebrafish inner ears driven by the agr2 promoter, to demonstrate and understand the mechanism by which the human CX26 R.184 mutation causes NSHL. Our results indicated that significant structural changes in the inner ears of transgenic lines with mutations were measured and compared to wild-type zebrafish. Simultaneously, significant alterations of transgenic lines with mutations in swimming behavior were analyzed with the zebrafish behavioral assay. This is the first study to investigate the functional results of the CX26 p.R184Q mutation with in vivo disease models. Our work supports and confirms the pathogenic role of the CX26 p.R184Q mutation in NSHL, with a hypothesized mechanism of altered interaction among amino acids in the connexins.

Enhancement of connexin30.3 expression in mouse embryonic stem cell line EB3 in response to cell-cell contacts.

To clarify the potential role of gap junction in cell-cell contact response, the expression of connexin30.3 gene (Cx30.3), a specifically expressed isoform in undifferentiated state of mouse embryonic stem (ES) cell line EB3 was investigated under different cell-cell contact conditions. ES cells were cultured by hanging drop culture method to increase cell-cell contact frequency. As control, a single cell culture was conducted. After culture for 12 h, the Cx30.3 expression level in hanging drop culture reached 1.73-fold that of the control (p < 0.001). By contrast, connexin43 gene (Cx43), a ubiquitously expressed gene, showed no difference between both cultures. The experiment of E-cadherin inhibition and ß-catenin knockdown suggested the action of E-cadherin upstream of the Cx30.3 regulating pathway. The cell-cell contacts with different cell lines such as HeLa cells and B16/BL6 caused no effect on the Cx30.3 in ES cells. These suggest a potential role of Cx30.3 as a cell-cell contact signal mediator partially regulated by E-cadherin signaling.

Erythrokeratodermia variabilis et progressiva.

Erythrokeratodermia variabilis et progressiva (EKVP) is a rare inherited skin disease characterized by fixed hyperkeratotic plaques and transient erythematous patches. EKVP is most often transmitted in an autosomal dominant manner. Causal mutations were found in the GJB3, GJB4 and GJA1 genes encoding connexins 31, 30.3 and 43, respectively. Approximately 50% of affected individuals develop palmoplantar keratoderma. Connexins are components of gap junctions, which are intercellular channels that are found in almost all tissues including the skin. Treatment of EKVP usually involves use of topical keratolytics and emollients resulting in some improvement in hyperkeratosis. Low-dose systemic retinoid may be beneficial. Novel therapies targeting connexin hemichannels and gap junctions may become available in the future.