Determination of structural features that underpin the pannexin1 channel inhibitory activity of the peptide 10Panx1.

Pannexin1 channels facilitate paracrine communication and are involved in a broad spectrum of diseases. Attempts to find appropriate pannexin1 channel inhibitors that showcase target-selective properties and in vivo applicability remain nonetheless scarce. However, a promising lead candidate, the ten amino acid long peptide mimetic 10Panx1 (H-Trp1-Arg2-Gln3-Ala4-Ala5-Phe6-Val7-Asp8-Ser9-Tyr10-OH), has shown potential as a pannexin1 channel inhibitor in both in vitro and in vivo studies. Nonetheless, structural optimization is critical for clinical use. One of the main hurdles to overcome along the optimization process consists of subduing the low biological stability (10Panx1 t1/2 = 2.27 ± 0.11 min). To tackle this issue, identification of important structural features within the decapeptide structure is warranted. For this reason, a structure-activity relationship study was performed to proteolytically stabilize the sequence. Through an Alanine scan, this study demonstrated that the side chains of Gln3 and Asp8 are crucial for 10Panx1's channel inhibitory capacity. Guided by plasma stability experiments, scissile amide bonds were identified and stabilized, while extracellular adenosine triphosphate release experiments, indicative of pannexin1 channel functionality, allowed to enhance the in vitro inhibitory capacity of 10Panx1.

Connexin 43 in Dermatofibroma and Dermatofibrosarcoma Protuberans: Diagnostic, Pathogenic, and Therapeutic Implications.

ABSTRACT: Connexins play a crucial role in the formation of gap junctions that connect cells to each other, as well as cells to the surrounding environment. In recent years, connexin 43 has been extensively studied in various human tumors. In this study, we conducted an immunohistochemical analysis to evaluate the expression of connexin in 16 dermatofibromas (DFs) and 13 dermatofibrosarcoma protuberans (DFSP). Connexin was diffusely expressed in the cytoplasm of all DFs with moderate or strong intensity, whereas all DFSPs showed negative staining. In addition to its diagnostic implications, the loss of Cx43 may elucidate the invasive capacity of DFSP and offer a potential avenue for future therapeutic interventions.

Connexin 43 Expression in Cutaneous Biopsies of Lupus Erythematosus.

INTRODUCTION: Gap junctions are channels between adjacent cells formed by connexins (Cxs). Cxs also form hemichannels that connect the cell with its extracellular milieu. These channels allow the transport of ions, metabolites, and small molecules; therefore, Cxs, and more specifically, connexin (Cx) 43 has been demonstrated to be in control of several crucial events such as inflammation and cell death. MATERIAL AND METHODS: We examined the immunostaining of Cx43 in the endothelia of the cutaneous blood vessels of biopsies from 28 patients with several variants of lupus erythematosus. RESULTS: In 19 cases (67.86%), staining of more than half of the dermal vessels including both vessels of the papillary and of the reticular dermis was identified. Only in 4 cases (14.28%), less than 25% of the vessels in the biopsy showed expression of the marker. CONCLUSIONS: Our results suggest a role of Cx43 in regulating the endothelial activity in lupus erythematosus, which also opens a door for targeted therapeutic options.

Expression of connexin 43 by atypical fibroxanthoma.

INTRODUCTION: Connexins are transmembrane channel proteins that interconnect adjacent cells and allow the exchange of signaling molecules between cells and the extracellular milieu. They have been investigated in many tumors to obtain information about tumor nature, behavior, and prognosis. METHODS: Herein, we present a study on the immunohistochemical expression of connexin (Cx) 43 in 16 cases of atypical fibroxanthoma (AFX). For the immunohistochemical staining, a tissue array was obtained from the paraffin-embedded blocks. RESULTS: The expression was membranous and cytoplasmic in all cases. Thirteen cases (81.25%) showed strong staining. In the other three cases (18.75%), the staining was medium. None of the cases showed nuclear staining. Fifteen out of 16 cases showed a diffuse pattern, and only one case showed a focal pattern. CONCLUSIONS: Our results suggest that Cx43 may play an important role in the natural behavior of AFX.

Walking together: women with the severe symptoms of menopause propose a platform for a walking program; outcome from focus groups.

BACKGROUND: Menopause and midlife are stages in a woman's life that can be marked by debilitating symptoms and increasing risks for cancer, cardiovascular, metabolic, and bone health issues. Walking represents a simple, low cost, and widely accessible activity with proven health benefits, though its therapeutic effect on alleviating menopause symptoms is not well characterized. Women are generally not opposed to exercise programs; however, increasing or maintaining exercise levels remains a challenge. We undertook a qualitative descriptive study to explore features of a walking program that would be conductive to menopausal women's participation, as well as to inform the development of such a program. METHODS: We conducted focus groups with women recruited from two menopause clinics and who suffered from moderate to severe menopause symptoms. The focus groups were audio recorded and transcribed. Women were prompted to talk about their menopause experience and exercise practice and how they would envision a walking exercise program that would keep them engaged. Qualitative content analysis was used to analyze the data and to identify characteristics of a walking exercise program. RESULTS: Twenty women participated in 5 focus groups. Women were very interested in trying walking as a means of staying healthy and possibly reducing menopause symptoms. Four major characteristics emerged as important for a walking program: (a) sensitivity to health realities of menopausal women, (b) inclusivity of various needs/levels of physical ability, (c) attentiveness to the need for mutual social support, (d) flexibility in planning of locations and scheduling. A restricted social network platform with features catering to women in menopause was suggested as suitable to initiate and sustain an adequate walking program. CONCLUSIONS: The findings of this study will be essential in designing a program that would be attractive for women to start and maintain a walking habit. The program would assist in elucidating whether walking is a useful and valuable alternative therapy for menopausal symptoms and, ultimately, might help women staying fit in midlife and postmenopausal.

Expression of Connexin 43 in 32 Cases of Merkel Cell Carcinoma.

INTRODUCTION: Connexins (Cxs) are channel proteins that allow direct connection among cells and between cells and the extracellular space. There is very little information in the literature on the expression of Cxs by Merkel cell carcinoma (MCC). MATERIALS AND METHODS: Thirty-two cases of MCC were recovered from our archives and studied immunohistochemically for Cx43. RESULTS: All our cases expressed several neuroendocrine markers. Most cases showed nonimmunohistochemically perceptible staining for Cx43. There was no difference between Merkel cell polyomavirus (MCPyV)-positive and MCPyV-negative cases. One case could not be evaluated. Only 2 cases showed a focal (10% of the tumor) membranous staining of Cx43. One of these cases was MCPyV-negative and, in the other, CM2B4 could not be evaluated. CM2B4 was positive in 18 cases and negative in 13 cases, and it could not be evaluated in 1 case. CONCLUSIONS: MCC shows a low Cx43 level, with no differences between MCPyV-positive and MCPyV-negative cases. Therefore, this opens the door for Cx43 targeting in therapeutic approaches to MCC.

Expression of Connexin 43 (Cx43) in Benign Cutaneous Tumors With Follicular Differentiation.

INTRODUCTION: Benign cutaneous tumors with follicular differentiation are alleged to differentiate toward parts of the hair follicle. Connexin 43 (Cx43) is a gap junction protein, the tumoral role of which has been investigated in several types of tumors. OBJECTIVE: To study the pattern of expression of Cx43 in benign cutaneous tumors with follicular differentiation and to compare it with that shown by their alleged anatomical counterparts of the hair follicle. MATERIALS AND METHODS: Five cases each of trichofolliculoma, trichilemmoma, fibrofolliculoma/trichodiscoma, trichoblastoma, trichoepithelioma, pilomatrixoma, and proliferating trichilemmal tumor, 3 cases of pilar sheath acanthoma, and 1 case of tumor of the follicular infundibulum were examined. Anti-Cx43 antibody was used. RESULTS: Cx43 was expressed by all follicular tumors studied. Comparisons between trichoblastoma and trichoepithelioma and their respective normal counterparts could not be made. In 3 tumors (trichofolliculoma, pilomatrixoma, and the spectrum fibrofolliculoma/trichodiscoma), there was a parallelism between their Cx43 expression pattern and that of their alleged anatomical counterparts. In pilar sheath acanthoma, trichilemmoma, and the tumor of the follicular infundibulum, we only found partial similarities in Cx43 expression. Only the proliferating trichilemmal tumor showed a discordant pattern of expression. CONCLUSIONS: Cx43 expression is preserved in benign cutaneous tumors with follicular differentiation and the patterns of Cx43 expression in benign cutaneous tumors with follicular differentiation parallel those of their alleged anatomical counterparts in 5 types (either totally or partially). This preservation might be related to the good behavior of the entities studied.

Recruitment of RNA molecules by connexin RNA-binding motifs: Implication in RNA and DNA transport through microvesicles and exosomes.

Connexins (Cxs) are integral membrane proteins that form high-conductance plasma membrane channels, allowing communication from cell to cell (via gap junctions) and from cells to the extracellular environment (via hemichannels). Initially described for their role in joining excitable cells (nerve and muscle), gap junctions (GJs) are found between virtually all cells in solid tissues and are essential for functional coordination by enabling the direct transfer of small signalling molecules, metabolites, ions, and electrical signals from cell to cell. Several studies have revealed diverse channel-independent functions of Cxs, which include the control of cell growth and tumourigenicity. Connexin43 (Cx43) is the most widespread Cx in the human body. The myriad roles of Cx43 and its implication in the development of disorders such as cancer, inflammation, osteoarthritis and Alzheimer's disease have given rise to many novel questions. Several RNA- and DNA-binding motifs were predicted in the Cx43 and Cx26 sequences using different computational methods. This review provides insights into new, ground-breaking functions of Cxs, highlighting important areas for future work such as transfer of genetic information through extracellular vesicles. We discuss the implication of potential RNA- and DNA-binding domains in the Cx43 and Cx26 sequences in the cellular communication and control of signalling pathways.

Expression of connexin 43 in the human hair follicle: emphasis on the connexin 43 protein levels in the bulge and through the keratinization process.

BACKGROUND: Gap junctions form communication compartments between cells. These channels assemble from connexin subunits. OBJECTIVE: To investigate the immunoexpression of connexin 43 (Cx43) in adult human hair follicles. METHODS: Cases were retrospectively obtained from our archives. RESULTS: We identified immunoexpression of Cx43 in the matrix, the papilla, the outer root sheath, the bulge, the medulla, the cortex, the shaft and the secretory part of the sebaceous gland. There was very low expression (VLE) of Cx43 in the perifollicular sheath, the mantle and the arrector pili muscle. The internal root sheath showed high-density expression in the bulb. Such expression abruptly decreased at different points in each of its layers at the point of keratinization. The isthmus showed Cx43-positive staining in the middle layers and all along, whereas there was VLE in the two outermost layers. The infundibulum showed expression all along the middle layers, whereas it showed VLE in the 2 outermost layers and in the 2 or 3 innermost layers. CONCLUSIONS: The bulge contains Cx43. Our results suggest that keratinization in the hair follicle is closely related to the decrease in Cx43 expression.

Nurturing maternal health in the midst of difficult life circumstances: a qualitative study of women and providers connected to a community-based perinatal program.

BACKGROUND: Many socioecological and structural factors affect women's diets, physical activity, and her access and receptivity to perinatal care. We sought to explore women's and providers' perceptions and experiences of health in the pre- and post-natal period while facing difficult life circumstances, and accessing a community-based program partially funded by Canada Prenatal Nutrition Program (CPNP) in Alberta, Canada. METHODS: Following the principles of community-based participatory research, we conducted a focused ethnography that involved five focus groups with women (28 in total), eight one-on-one interviews with program providers, and observations of program activities. Data were analyzed through qualitative content analysis to inductively derive codes and categories. RESULTS: Women perceived eating healthy foods, taking prenatal vitamins, and being physically active as key health behaviours during pregnancy and postpartum. However, they were commonly coping with many difficult life circumstances, and faced health barriers for themselves and their babies. These barriers included pregnancy or birth complications, family and spousal issues, financial difficulties, and living rurally. On the other hand, women and providers identified many aspects of the community-based program that addressed the burden of adversities as enablers to better health during pregnancy and postpartum. CONCLUSION: Community-based programs have an important role in alleviating some of the burden of coping with difficult life circumstances for women. With such potential, community-based programs need to be well supported through policies. Policies supporting these programs, and ensuring adequate funding, can enable more equitable services to rural women and truly promote maternal health during pregnancy and postpartum.

"A complex interface: Exploring sickle cell disease from a parent's perspective, after moving from Sub-Saharan Africa to North America".

INTRODUCTION: Sickle cell disease (SCD) is an inherited, multi-system, chronic disease with the highest prevalence affecting people of Sub-Saharan African descent. While major advances in SCD care have occurred over the last few decades in many African countries these advances are not readily available. Prior literature from Ghana and Kenya describe stigma, despair, and economic burden as well as hope when a child has SCD. When people migrate to North America with a child with SCD it is unknown whether their perception of the disease changes. We asked, "How do immigrant parents of children with SCD from Sub-Saharan Africa perceive, and manage the disease in the context of western medical care?" METHODS: The research question was explored with qualitative methodology, specifically focused ethnography. Semi-structured interviews were conducted with parent(s). The interviews were audio recorded, transcribed, and open coded. Rigor was determined through methodological coherence, appropriate and sufficient sampling, and iterative data collection and analysis. RESULTS: Twelve interviews were conducted. Identified themes are as follows: memories of SCD in Africa, the emotional journey towards acceptance, and parental approach to care for their child. CONCLUSIONS: Healthcare providers should be responsive to an immigrant families' needs and not expect linear progression of emotional acceptance to the diagnosis. Healthcare providers patience with the process helps establish trust, works to facilitate and encourage hope and acknowledges the strength of the families, and their dedication to their family member. Healthcare providers should acknowledge parents' sources of support (religion/family) and ensure parents are aware of medical advances.

Peyakohewamak-Needs of Involved Nehiyaw (Cree) Fathers Supporting Their Partners During Pregnancy: Findings From the ENRICH Study.

We sought to understand the needs of involved Nehiyaw (Cree) fathers who supported their partners during pregnancy. We used qualitative description and a community-based participatory research approach. We carried out in-depth semi-structured interviews with six Nehiyaw fathers. Four also participated in photovoice and follow-up interviews. All data were content analyzed qualitatively. Fathers felt they had to support their partners and overcome challenges resulting from intergenerational colonial impacts (residential schools particularly) by reclaiming their roles and acknowledging the pregnancy as a positive change. Providing support was possible through their own strong support system stemming from family, faith, culture, and a stable upbringing with positive male role models and intact Nehiyaw kinships. Perinatal programming did little to include fathers. Attempts to improve perinatal care and outcomes should allow more inclusion of and support for Indigenous fathers through genuinely incorporating into care traditional culture and Elders, families, flexibility, cultural understanding, and reconciliation.

The determinants of meaning of pain following an acute traumatic injury.

RATIONALE AND AIMS: Meaning is an integral aspect of life that drives behaviours, actions and emotions. Perception of pain is believed to be affected by the meaning of pain. Our primary aim was to investigate and discuss the determinants of meaning in acute pain following a traumatic injury. METHODOLOGICAL DESIGN: Using the Interpretive Description approach, a method of qualitative inquiry, 13 adults hospitalised due to their accidental injuries were recruited. Semi-structured interviews that were digitally recorded were used to collect the data. Ethical approval was received by our local Health Research Ethics Board, and all relevant ethical standards were followed as outlined in the approved ethics proposal. FINDINGS: The three primary determinants of meaning during an acute pain event, or the experience of acute pain include permanence of injuries, incongruence of care quality, and personal responses to the injury and care received. Although the permanence of an injury is lasting, we did not find any emotional response to this fact while the participants were hospitalised. The emotion characterising the personal response to the perceived poor quality of hospital care received was anxiety. CONCLUSION: Both the technical and personal components of healthcare quality have the potential to increase the perception of pain. Meanings in an acute pain event are primarily related to the determinants of incongruent care and the personal response to that determinant: anxiety.

Women's perceptions of discussions about gestational weight gain with health care providers during pregnancy and postpartum: a qualitative study.

BACKGROUND: Maternal body weight is an indicator of the health of a mother and her developing fetus. Risks of poor maternal and fetal health issues increase when women gain too little or too much weight during pregnancy. A study of 600 women from Alberta, Canada, reported approximately 30, 46, 80, and 80% of underweight, healthy weight, overweight, and obese women, respectively, gained in excess of Health Canada gestational weight gain guidelines. Behavioural interventions during pregnancy have shown to be effective at supporting women achieve gestational weight gain (GWG) recommendations and return to their pre-pregnancy weight postpartum, yet few women are counseled about weight gain during pregnancy. A discrepancy exists between health care providers' (HCP) reported counseling behaviours and women's perceptions of counseling by HCPs; most HCPs report counseling women about GWG; conversely, most women report not receiving counseling about GWG. This study explored women's experiences with GWG and their perceptions of discussions about GWG with HCPs during pregnancy and postpartum. This will help to identify gaps in service delivery and highlight areas for improvement that may better support women to achieve GWG recommendations leading to better health outcomes for women and children. METHODS: Five focus groups (n = 26) were conducted with women up to 1 year postpartum across the five Alberta health zones. Focus groups were transcribed verbatim and analyzed using qualitative content analysis. RESULTS: GWG is important to women, for their health and for the health of their baby. In-depth conversations with HCPs about GWG or weight loss do not occur; however, women want the opportunity to discuss weight gain/loss with HCPs. Women would like discussions about gestational weight gain/loss to become part of standard care and offered to all women. CONCLUSIONS: Women suggested that discussions about GWG should occur with all women, and that HCPs should initiate these discussions by asking women how they feel about discussing weight. Conversations should begin early on in pregnancy and continue through to the postpartum period. Interventions assessing discussions about GWG should be implemented and evaluated as this has been identified as a gap in prenatal service delivery.

Proteomic Analysis of Connexin 43 Reveals Novel Interactors Related to Osteoarthritis.

We have previously reported that articular chondrocytes in tissue contain long cytoplasmic arms that physically connect two distant cells. Cell-to-cell communication occurs through connexin channels termed Gap Junction (GJ) channels, which achieve direct cellular communication by allowing the intercellular exchange of ions, small RNAs, nutrients, and second messengers. The Cx43 protein is overexpressed in several human diseases and inflammation processes and in articular cartilage from patients with osteoarthritis (OA). An increase in the level of Cx43 is known to alter gene expression, cell signaling, growth, and cell proliferation. The interaction of proteins with the C-terminal tail of connexin 43 (Cx43) directly modulates GJ-dependent and -independent functions. Here, we describe the isolation of Cx43 complexes using mild extraction conditions and immunoaffinity purification. Cx43 complexes were extracted from human primary articular chondrocytes isolated from healthy donors and patients with OA. The proteomic content of the native complexes was determined using LC-MS/MS, and protein associations with Cx43 were validated using Western blot and immunolocalization experiments. We identified >100 Cx43-associated proteins including previously uncharacterized proteins related to nucleolar functions, RNA transport, and translation. We also identified several proteins involved in human diseases, cartilage structure, and OA as novel functional Cx43 interactors, which emphasized the importance of Cx43 in the normal physiology and structural and functional integrity of chondrocytes and articular cartilage. Gene Ontology (GO) terms of the proteins identified in the OA samples showed an enrichment of Cx43-interactors related to cell adhesion, calmodulin binding, the nucleolus, and the cytoskeleton in OA samples compared with healthy samples. However, the mitochondrial proteins SOD2 and ATP5J2 were identified only in samples from healthy donors. The identification of Cx43 interactors will provide clues to the functions of Cx43 in human cells and its roles in the development of several diseases, including OA.

Qualitative Assessment of Key Messages about Nutrition and Weight Gain in Pregnancy in Printed Educational Materials in Alberta.

Printed educational materials are a common source of health information, although their effectiveness in improving women's knowledge or self-care in pregnancy has been questioned. This study describes the information in printed educational materials that address healthy eating during pregnancy and gestational weight gain (GWG) that are currently used in Alberta, Canada. Content of 6 resources was analyzed using a constant comparison qualitative approach. Resources emphasized healthy eating, prenatal supplements, folate supplementation, and healthy weight gain. More resources discussed the importance of "eating enough" than provided guidance on avoiding excessive GWG. Themes identified were: "everything is important" meaning that all healthy behaviours are important, making prioritization difficult; "more is more" emphasized eating more over moderation; "everyone is individual" suggests women seek individualized care through the care provider; and "contradictions" describes differences in content and recommendations within and between resources. New or revised versions of resources should provide congruent information with up-to-date recommendations that are easily prioritized. Care providers should be aware of contradictory information or information that does not align with current recommendations within printed educational materials and be ready to help women address the areas important for her personal behaviour change.

Contrasting "back home" and "here": how Northeast African migrant women perceive and experience health during pregnancy and postpartum in Canada.

BACKGROUND: International migration and the number of migrant women who experience pregnancy and childbirth in receiving countries have significantly increased in the last two decades. Migrant women often have unmet social and economic needs during pregnancy, and are more likely to have problems unaddressed by health care systems. In this qualitative study, we explored migrant women's perceptions and experiences of health during pregnancy and postpartum, while participating in a perinatal program offered through a community-based organization. Additionally, we examined sociocultural factors that might have shaped women's health upon migration to the Canadian city of Edmonton, Alberta. METHODS: A community-based participatory research approach was used to engage migrant women connected to a community-based perinatal program in Edmonton. A focused ethnography was conducted with four Northeast African communities (Eritrean, Ethiopian, Oromo and Somali), and involved 10 focus groups with women (n = 8, per group) and direct observations of weekly perinatal program activities. Data generation and analysis occurred concurrently, and all generated data were analyzed using qualitative content analysis to inductively derive codes and categories. RESULTS: Women expressed their perceptions and experiences of health during pregnancy and postpartum by contrasting their countries of origin with Canada, respectively identified as "back home" and "here". Differences in social support and the physical environment (both natural and built) between "back home" and "here" were commonly described as factors that shaped their opportunities to eat healthy, be physically active and emotionally well before and after having a baby "here". Overall, women described that in Canada they lacked the social and environmental factors perceived as key enablers of healthy pregnancies and postpartum. CONCLUSION: A complex network of factors seem to influence Northeast African women's health during pregnancy and postpartum upon migration to Canada. It is of the utmost importance to provide these women with the immediate sociocultural and environmental factors they need to successfully thrive during pregnancy and postpartum, especially while establishing social and support networks "here".

Kikiskawâwasow - prenatal healthcare provider perceptions of effective care for First Nations women: an ethnographic community-based participatory research study.

BACKGROUND: Pregnant Indigenous women suffer a disproportionate burden of risk and adverse outcomes relative to non-Indigenous women. Although there has been a call for improved prenatal care, examples are scarce. Therefore, we explored the characteristics of effective care with First Nations women from the perspective of prenatal healthcare providers (HCPs). METHODS: We conducted an ethnographic community-based participatory research study in collaboration with a large Cree First Nations community in Alberta, Canada. We carried out semi-structured interviews with 12 prenatal healthcare providers (HCPs) that were recorded, transcribed, and subjected to qualitative content analysis. RESULTS: According to the participants, relationships and trust, cultural understanding, and context-specific care were key features of effective prenatal care and challenge the typical healthcare model. HCPs that are able to foster sincere, non-judgmental, and enjoyable interactions with patients may be more effective in treating pregnant First Nations women, and better able to express empathy and understanding. Ongoing HCP cultural understanding specific to the community served is crucial to trusting relationships, and arises from real experiences and learning from patients over and above relying only on formal cultural sensitivity training. Consequently, HCPs report being better able to adapt a more flexible, all-inclusive, and accessible approach that meets specific needs of patients. CONCLUSIONS: Aligned with the recommendations of the Truth and Reconciliation Commission of Canada, improving prenatal care for First Nations women needs to allow for genuine relationship building with patients, with enhanced and authentic cultural understanding by HCPs, and care approaches tailored to women's needs, culture, and context.

Worth the Risk? Muddled Relationships in Community-Based Participatory Research.

Community-based participatory research (CBPR) is a collaborative research approach that has two purposes: (a) to generate knowledge about and (b) to take action to improve the lives of people facing health, social, economic, political, and environmental inequities. The foundation of all CBPR projects is its partnership--its cooperative relationship between community members, service providers, program planners, policy makers, and academics. It is with people--and through relationships--that partnerships are built and sustained. Although relationships between academics and community members are critical to creating knowledge and change, they are overlooked in the literature. We often hear about CBPR "gone wrong," when tensions and conflicts arise because relationship boundaries become blurred. Our purpose is to expose the muddled relationships that can be created between academics and community members in CBPR projects. Drawing upon our experiences presented in a series of vignettes, we consider the nature of these relationships. We explore whether we conduct, in CBPR, good research at the expense of muddling relationships. Despite the potential for muddled relationships, we believe that CBPR is the best approach for research aimed at achieving a more equitable and just society.

Articular chondrocyte network mediated by gap junctions: role in metabolic cartilage homeostasis.

OBJECTIVE: This study investigated whether chondrocytes within the cartilage matrix have the capacity to communicate through intercellular connections mediated by voltage-gated gap junction (GJ) channels. METHODS: Frozen cartilage samples were used for immunofluorescence and immunohistochemistry assays. Samples were embedded in cacodylate buffer before dehydration for scanning electron microscopy. Co-immunoprecipitation experiments and mass spectrometry (MS) were performed to identify proteins that interact with the C-terminal end of Cx43. GJ communication was studied through in situ electroporation, electrophysiology and dye injection experiments. A transwell layered culture system and MS were used to identify and quantify transferred amino acids. RESULTS: Microscopic images revealed the presence of multiple cellular projections connecting chondrocytes within the matrix. These projections were between 5 and 150 µm in length. MS data analysis indicated that the C-terminus of Cx43 interacts with several cytoskeletal proteins implicated in Cx trafficking and GJ assembly, including α-tubulin and ß-tubulin, actin, and vinculin. Electrophysiology experiments demonstrated that 12-mer oligonucleotides could be transferred between chondrocytes within 12 min after injection. Glucose was homogeneously distributed within 22 and 35 min. No transfer was detected when glucose was electroporated into A549 cells, which have no GJs. Transwell layered culture systems coupled with MS analysis revealed connexins can mediate the transfer of L-lysine and L-arginine between chondrocytes. CONCLUSIONS: This study reveals that intercellular connections between chondrocytes contain GJs that play a key role in cell-cell communication and a metabolic function by exchange of nutrients including glucose and essential amino acids. A three-dimensional cellular network mediated through GJs might mediate metabolic and physiological homeostasis to maintain cartilage tissue.