Patricia E. Martin

Connexin mimetic peptides improve cell migration rates of human epidermal keratinocytes and dermal fibroblasts in vitro

Nonhealing cutaneous wounds, a major cause of morbidity and mortality, are difficult to treat. Recent studies suggest that significant increases in skin wound‐healing rates occur by altering gap junction intercellular communication (GJIC). As migration of keratinocytes and fibroblasts is an important feature of wound healing, this study investigated whether migration rates in cultured normal human epidermal keratinocytes and dermal fibroblasts could be altered by modulating GJIC via connexin mimetic peptides. First, HeLa cells stably transfected with connexin43 (Cx43), Cx40, or Cx26 were used as a model to determine connexin specificity and the doses of connexin mimetic peptides required to attenuate GJIC. Gap26 and Gap26M inhibited GJIC dose dependently and were nonconnexin specific, whereas Gap27 was Cx43‐selective. Skin keratinocytes and fibroblasts expressed a variety of connexins, with Cx43 predominating. Cx43 protein expression was reduced at leading edges 3 hours after scraping confluent monolayers, resolving at 24 hours. Gap26M and Gap27 significantly increased migration rates across scrapes in keratinocytes and fibroblasts by blocking gap junction functionality. GJIC inhibition can thus directly influence keratinocyte and fibroblast migration. Furthermore, our results support the therapeutic potential of connexin mimetic peptides to aid wound closure, and provide a simple approach to screening new agents.

Effects of connexin-mimetic peptides on gap junction functionality and connexin expression in cultured vascular cells

1 We have investigated the effects of connexin‐mimetic peptides homologous to the Gap 26 and Gap 27 domains of Cxs 37, 40 and 43 against gap junctional communication and connexin expression in rat aortic endothelial cells (RAECs) and A7r5 myocytes. 2 Immunostaining and Western blot analysis confirmed the presence of gap junction plaques containing Cx43, but not Cx40, in RAECs, whereas plaques containing Cxs 40 and 43 were evident in A7r5 cells. Expression of Cx37 was limited in RAECs and absent from A7r5 cells. 3 Under control conditions calcein‐loaded RAECs transferred dye to ∼70% of subjacent A7r5 cells after coculture for 4–5 h. Dye transfer was inhibited by a peptide targeted to Cxs 37 and 43 (37,43Gap 27), but minimally affected by peptides targeted to Cxs 37 and 40 (37,40Gap 26 and 40Gap 27). These findings suggest that the myoendothelial gap junctions that couple RAECs and A7r5 cells are constructed principally from Cx43. 4 Inhibition of dye transfer from RAECs to A7r5 cells cocultured in the presence of 37,43Gap 27 plus 37,40Gap 26 for 5 h was fully reversible. 5 In A7r5 cells, endogenous expression of Cx40 and Cx43 was unaffected by incubation with 37,43Gap 27, 37,40Gap 26, either individually or in combination, and the peptide combination did not impair connexin trafficking or the de novo formation of gap plaques in A7r5 cells transfected to express Cx43‐GFP. 6 Treatment of A7r5 cells with 37,43Gap 27 plus 37,40Gap 26 abolished synchronized oscillations in intracellular [Ca2+] induced by the α1‐adrenoceptor agonist phenylephrine. 7 The reversibility and lack of effect of the peptides on plaque formation suggests that they may be considered ideal probes for functional studies of connexin‐mediated communication in the vascular wall.

Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non-diabetic cells

During early wound healing (WH) events Connexin 43 (Cx43) is down‐regulated at wound margins. In chronic wound margins, including diabetic wounds, Cx43 expression is enhanced suggesting that down‐regulation is important for WH. We previously reported that the Cx43 mimetic peptide Gap27 blocks Cx43 mediated intercellular communication and promotes skin cell migration of infant cells in vitro. In the present work we further investigated the molecular mechanism of Gap27 action and its therapeutic potential to improve WH in skin tissue and diabetic and non‐diabetic cells. Ex vivo skin, organotypic models and human keratinocytes/fibroblasts of young and old donors and of diabetic and non‐diabetic origin were used to assess the impact of Gap27 on cell migration, proliferation, Cx43 expression, localization, phosphorylation and hemichannel function. Exposure of ex vivo WH models to Gap27 decreased dye spread, accelerated WH and elevated cell proliferation. In non‐diabetic cell cultures Gap27 decreased dye uptake through Cx hemichannels and after scratch wounding cells showed enhanced migration and proliferation. Cells of diabetic origin were less susceptible to Gap27 during early passages. In late passages these cells showed responses comparable to non‐diabetic cells. The cause of the discrepancy between diabetic and non‐diabetic cells correlated with decreased Cx hemichannel activity in diabetic cells but excluded differences in Cx43 expression, localization and Ser368‐phosphorylation. These data emphasize the importance of Cx43 in WH and support the concept that Gap27 could be a beneficial therapeutic to accelerate normal WH. However, its use in diabetic WH may be restricted and our results highlight differences in the role of Cx43 in skin cells of different origin.

Reversible Connexin 43 Dephosphorylation During Hypoxia and Reoxygenation Is Linked to Cellular ATP Levels

Altered gap junction coupling of cardiac myocytes during ischemia may contribute to development of lethal arrhythmias. The phosphoprotein connexin 43 (Cx43) is the major constituent of gap junctions. Dephosphorylation of Cx43 and uncoupling of gap junctions occur during ischemia, but the significance of Cx43 phosphorylation in this setting is unknown. Here we show that Cx43 dephosphorylation in synchronously contracting myocytes during ischemia is reversible, independent of hypoxia, and closely associated with cellular ATP levels. Cx43 became profoundly dephosphorylated during hypoxia only when glucose supplies were limited and was completely rephosphorylated within 30 minutes of reoxygenation. Similarly, direct reduction of ATP by various combinations of metabolic inhibitors and by ouabain was closely paralleled by loss of phosphoCx43 and recovery of phosphoCx43 accompanied restoration of ATP. Dephosphorylation of Cx43 could not be attributed to hypoxia, acid pH or secreted metabolites, or to AMP-activated protein kinase; moreover, the process was selective for Cx43 because levels of phospho-extracellular signal regulated kinase (ERK)1/2 were increased throughout. Rephosphorylation of Cx43 was not dependent on new protein synthesis, or on activation of protein kinases A or G, ERK1/2, p38 mitogen-activated protein kinase, or Jun kinase; however, broad-spectrum protein kinase C inhibitors prevented Cx43 rephosphorylation while also sensitizing myocytes to reoxygenation-mediated cell death. We conclude that Cx43 is reversibly dephosphorylated and rephosphorylated during hypoxia and reoxygenation by a novel mechanism that is sensitive to nonlethal fluctuations in cellular ATP. The role of this regulated phosphorylation in the adaptation to ischemia remains to be determined.

ATP release by cardiac myocytes in a simulated ischaemia model Inhibition by a connexin mimetic and enhancement by an antiarrhythmic peptide

We studied the role of connexin hemichannels in the release of ATP by neonatal cardiac myocytes subject to ischaemic stress. Mechanical, osmotic and oxidative stress and changes in extracellular or intracellular Ca(2+) levels induce connexin hemichannels located in the plasma membrane to open and release small ions and molecules with signaling potential such as ATP. Since ATP release has been implicated in adaptation to oxygen deprivation, we studied its release by cardiac myocytes incubated in a custom-built hypoxia chamber for various periods. In a simulated ischaemia model (0.5% oxygen and 0.2 g/l glucose) a peak of ATP release occurred at 80 min followed by a return to steady state levels for a further 200 min. This peak of ATP release was not observed in myocytes subject to hypoxia (0.5% oxygen, 3.0 g/l glucose). ATP release in ischaemia was influenced by two classes of reagents that target connexins, the channel forming proteins of gap junctions. First, the connexin hemichannel inhibitors Gap 26 and 18a glycyrrhetinic acid abolished the ATP peak of release. Second, the AAP10, a peptide with antiarrhythmic properties markedly increased the peak of ATP release observed at 80 min of ischaemia and also induced a second smaller peak at 180-240 min. ATP content of the myocytes and Cx43 phosphorylation were monitored. Since the release of ATP in ischaemia was abolished by connexin channel inhibitors and stimulated by a peptide developed to target connexins in the context of cardiac arrhythmia, the results suggest that nucleotide release by connexin hemichannels is likely to feature in the response of myocytes to ischaemic stress in the heart.

The antiarrhythmic peptide rotigaptide (ZP123) increases gap junction intercellular communication in cardiac myocytes and HeLa cells expressing connexin 43

We investigated the effects of rotigaptide (ZP123), a stable hexapeptide with antiarrhythmic properties, on gap junction mediated intercellular communication in contracting rat neonatal cardiac myocytes, HL‐1 cells derived from cardiac atrium and in HeLa cells transfected with cDNA encoding Cx43‐GFP, Cx32‐GFP, Cx26‐GFP, wild‐type Cx43 or wild‐type Cx26. Intercellular communication was monitored before and after treatment with rotigaptide following microinjection of small fluorescent dyes (MW<1 kDa). The communication‐modifying effect of rotigaptide was confined to cells expressing Cx43 since the peptide had no effect on dye transfer in HeLa cells expressing Cx32‐GFP, Cx26‐GFP or wild‐type Cx26. In contrast, HeLa cells expressing Cx43‐GFP exposed to 50 nM rotigaptide for 5 h showed a 40% increase in gap junction mediated communication. Rotigaptide (50 nM) increased intercellular dye transfer in myocytes and atrial HL‐1 cells, where Cx43 is the dominant connexin. However, it caused no change in cell beating rates of cardiac myocytes. Western blot analysis showed that rotigaptide did not modify the overall level of Cx43 expression and changes in the phosphorylation status of the protein were not observed. We conclude that the effects of rotigaptide were confined to cells expressing Cx43.

A novel missense mutation in GJB2 disturbs gap junction protein transport and causes focal palmoplantar keratoderma with deafness

Gap junctions are intercellular channels that mediate rapid intercellular communication. They consist of connexins, small transmembrane proteins that belong to a large family found throughout the animal kingdom. In the skin, several connexins are expressed and are involved in the regulation of epidermal growth and differentiation. One of the skin expressed gap junction genes is GJB2, which codes for connexin 26 and is associated with a wide variety of keratinisation disorders. Here, we report on a family with a novel GJB2 mutation (p.His73Arg) causing a syndrome of focal palmoplantar keratoderma with severe progressive sensorineural hearing impairment, a phenotype reminiscent of Vohwinkel syndrome. Using fluorescent connexin fusion proteins, we show that the mutation induces a transport defect similar to that found for the Vohwinkel syndrome mutation p.Asp66His. Co-transfection into cells expressing wild type connexin26 shows that the mutant has a dominant negative effect on connexin trafficking. We suggest that there may be a weak genotype–phenotype correlation for mutations in GJB2.

Peptidoglycan derived from Staphylococcus epidermidis induces Connexin43 hemichannel activity with consequences on the innate immune response in endothelial cells

Gram-positive bacterial cell wall components including PGN (peptidoglycan) elicit a potent pro-inflammatory response in diverse cell types, including endothelial cells, by activating TLR2 (Toll-like receptor 2) signalling. The functional integrity of the endothelium is under the influence of a network of gap junction intercellular communication channels composed of Cxs (connexins) that also form hemichannels, signalling conduits that are implicated in ATP release and purinergic signalling. PGN modulates Cx expression in a variety of cell types, yet effects in endothelial cells remain unresolved. Using the endothelial cell line b.End5, a 6 h challenge with PGN induced IL-6 (interleukin 6), TLR2 and Cx43 mRNA expression that was associated with enhanced Cx43 protein expression and gap junction coupling. Cx43 hemichannel activity, measured by ATP release from the cells, was induced following 15 min of exposure to PGN. Inhibition of hemichannel activity with carbenoxolone or apyrase prevented induction of IL-6 and TLR2 mRNA expression by PGN, but had no effect on Cx43 mRNA expression levels. In contrast, knockdown of TLR2 expression had no effect on PGN-induced hemichannel activity, but reduced the level of TLR2 and Cx43 mRNA expression following 6 h of PGN challenge. PGN also acutely induced hemichannel activity in HeLa cells transfected to express Cx43, but had no effect in Cx43-deficient HeLa OHIO cells. All ATP responses were blocked with Cx-specific channel blockers. We conclude that acute Cx43 hemichannel signalling plays a role in the initiation of early innate immune responses in the endothelium.

Porokeratotic eccrine nevus may be caused by somatic connexin26 mutations.

Porokeratotic eccrine ostial and dermal duct nevus, or porokeratotic eccrine nevus (PEN), is a hyperkeratotic epidermal nevus. Several cases of widespread involvement have been reported, including one in association with the keratitis–ichthyosis–deafness (KID) syndrome (OMIM #148210), a rare disorder caused by mutations in the GJB2 gene coding for the gap junction protein connexin26 (Cx26). The molecular cause is, as yet, unknown. We have noted that PEN histopathology is shared by KID. The clinical appearance of PEN can resemble that of KID syndrome. Furthermore, a recent report of cutaneous mosaicism for a GJB2 mutation associated with KID describes linear hyperkeratotic skin lesions that might be consistent with PEN. From this, we hypothesized that PEN might be caused by Cx26 mutations associated with KID or similar gap junction disorders. Thus, we analyzed the GJB2 gene in skin samples from two patients referred with generalized PEN. In both, we found GJB2 mutations in the PEN lesions but not in unaffected skin or peripheral blood. One mutation was already known to cause the KID syndrome, and the other had not been previously associated with skin symptoms. We provide extensive functional data to support its pathogenicity. We conclude that PEN may be caused by mosaic GJB2 mutations.

The connexin mimetic peptide Gap27 increases human dermal fibroblast migration in hyperglycemic and hyperinsulinemic conditions in vitro

Significant increases in skin wound healing rates occur by reducing connexin‐mediated communication (CMC). Gap27, a connexin (Cx) mimetic peptide targeted to the second extracellular loop of Cx43, which inhibits CMC, increases migration of human keratinocytes and dermal fibroblasts. To examine the efficacy of Gap27 in a hyperglycemic and hyperinsulinemic in vitro environment, cell migration, gap junction, and Cx hemichannel functionality and cell–substrate adhesion assays were performed on human dermal fibroblasts and diabetic fibroblast and keratinocytes. To investigate fibroblast genes involved in these processes, extra‐cellular matrix (ECM) and adhesion gene expression was determined with a PCR array. Gap27 increased fibroblast migration in both euglycemia/euinsulinemia and hyperglycemia/hyperinsulinemia, and influenced migration in diabetic keratinocytes. Hyperglycemia/hyperinsulinemia reduced gap junction coupling in fibroblasts and Gap27 reduced CMC and cell adhesion to substrata in fibroblasts cultured in high glucose. Migrating dermal fibroblast ECM and cell adhesion genes were found to be differentially regulated by Gap27 in euglycemia and hyperglycemia. The PCR array showed that Gap27 upregulated 34 genes and downregulated 1 gene in euglycemic migrating fibroblasts. By contrast in hyperglycemia, Gap27 upregulated 1 gene and downregulated 9 genes. In euglycemic conditions, Gap27 induced upregulation of genes associated with ECM remodeling, whereas in hyperglycemia, ECM component genes were downregulated by Gap27. Thus, Gap27 improves cell migration during scrape‐wound repair in hyperglycemia/hyperinsulinemia conditions in vitro, although migration of diabetic cells is less influenced. Our results suggest that this increase in motility may occur by decreasing gap junction and hemichannel activity and altering gene expression in the adhesion and ECM pathway. J. Cell. Physiol. 227: 77–87, 2012.