Connexin 43, Bcl-2, Bax, Ki67, and E-cadherin patterns in oral squamous cell carcinoma and its relationship with GJA1 rs12197797 C/G

Background: To our knowledge, there is no useful and accurate prognostic biomarker or biomarkers for patientswith oral squamous cell carcinoma (OSCC), a tumor with uncertain biological behavior, and unpredictable clinical progress. The purposes of this study were: a) to determine the expresión profile of Connexin 43, Bcl-2, Bax,E-cadherin, and Ki67 in patients with OSCC; b) identify the GJCA1 rs12197797 genotypic composition.Material and Methods: A cross-sectional study using genomic DNA and biopsy samples extracted from the oralmucosa with/without OSCC, older than 18 years, both genders, attended at Facultad de Odontología, UniversidadNacional Córdoba. Immunostaining for Cx43, Bcl-2, Bax, E-cadherin, and Ki67 and genotyping GJA1 rs12197797by RFLP were performed. Odds Ratio (95% CI), Spearman Coefficient were estimated. Mann-Whitney test wasapplied to analyze immunostaining between controls/cases (p <0.05 was set for statistical significance).Results: GG (mutant) was the most frequent genotype in patients with OSCC diagnosis (53.2%) in relation toCC “healthy” genotype (p=0.00487; OR=7.33; CI95% [1.1-54.7]). And, the allele G (mutant) had a presence in75.5% of OSCC patients. However, no significant association was observed between alleles C/G and diagnosis(p=0.0565). The heterozygous genotype was the most frequent in the patients of both groups Cx43 and E-cadherinmarkers were lower in OSCCs in relation to controls. Ki67 and Bcl-2 immunolabeling were high on OSCC, andBax immunomarker was diminished in OSCC.Conclusions: We hypothesized that the oral epithelium losses Connexin 43 and E-cadherin in the membrane, whichmodifies cell differentiation. The Ki67 and Bcl2 overexpression would increase the cell density in the tissue, by promoting proliferation and decreasing apoptosis. And, this study shows evidence that patients who carry on allele G ofGJA1rs12197797 could be at risk of developing OSCC. (AU)

Implicación de la Cx43 y el cilio primario en la actividad de los osteocitos / Cx43 and primary cilium involvement in osteocyte activity

Objetivo: El tejido óseo tiene la capacidad de adaptarse a los estímulos del entorno alterando su morfología y metabolismo. Las diferentes células óseas se comunican entre sí a través de uniones comunicantes (UCs). La conexina 43 (Cx43) es la proteína más abundante de las UCs; tiene funciones clave en la transducción de señales y en la respuesta a estímulos hormonales y mecánicos. Otro elemento mecanosensor de los osteocitos es el cilio primario, formado por microtúbulos y que se desarrolla en la fase G0 del ciclo celular. Los objetivos de este estudio fueron determinar la implicación de la Cx43 y del cilio primario en la actividad de los osteocitos, analizar la posible interacción entre estos dos mecanosensores, y evaluar el papel que desempeñan en la detección y respuesta de los osteocitos ante el estímulo mecánico y la estimulación del receptor de la parathormona tipo 1 (PTH1R) por su ligando, la proteína relacionada con la parathormona (PTHrP) (1-36). Material y métodos: Se comparó la línea celular de osteocitos MLO-Y4 control (Cx43+/+) con MLO-Y4 deficientes en Cx43 (Cx43-/-). El análisis de expresión de la proteína del transporte intraflagelar 88 (IFT88), de la Cx43 y de la fosforilación de la quinasa reguladora de la señal extracelular (P-ERK) se determinó mediante Western blot. Para caracterizar la posible colocalización entre el cilio primario y Cx43 se realizó una inmunofluorescencia. Para simular el estímulo mecánico in vitro, las células se sometieron a un estrés mecánico de 10 dinas/cm2 por flujo de fluido durante 10 minutos. Resultados: Los resultados obtenidos muestran que el número de células con cilio primario no varía por la expresión de Cx43 (p=0,089); y que en las células con presencia en Cx43, el estímulo mecánico por flujo de fluido y la PTHrP aumentan la fosforilación de quinasas reguladas por señal extracelular (ERK) respecto a las células no estimuladas (p=0,049 y p=0,011, respectivamente). (AU)

Objetivo: Bone tissue can adapt to environmental stimuli by altering its morphology and metabolism. Different bone cells communicate with each other through communicating junctions (CJs). Connexin 43 (Cx43) is the most abundant CJ protein with key functions in signal transduction and in response to hormonal and mechanical stimuli. Another mechanosensor element of osteocytes is the primary cilium, formed by microtubules and which develops in the cell cycle’s G0 phase. Our study aims to determine Cx43 and primary cilium involvement in osteocytic activity, to analyze the possible interaction between these two mechanosensors and to assess the role they play in the detection and response of osteocytes to mechanical stimuli and stimulation of the parathormone type 1 receptor (PTH1R) by its ligand, the parathormone-related protein (PTHrP) (1-36). Material and methods: The control MLO-Y4 (Cx43 +/+) osteocyte cell line was compared to Cx43-deficient MLO-Y4 (Cx43 -/-). The expression analysis of intraflagellar transport protein 88 (IFT88), Cx43 and phosphorylation of the extracellular signal regulatory kinase (P-ERK) was determined by Western blot. To characterize the possible colocalization between the primary cilium and Cx43, an immunofluorescence was carried out. To simulate mechanical stimulation in vitro, cells were subjected to mechanical stress of 10 dynes/cm2 by fluid flow for 10 minutes. Results: The results obtained show that the number of cells with primary cilium does not vary due to the expression of Cx43 (p = 0.089). In cells with Cx43 presence, mechanical stimulation by fluid flow and PTHrP increase the phosphorylation of extracellular signal-regulated kinases (ERK) compared to unstimulated cells (p = 0.049 and p = 0.011, respectively). (AU)

Regulación de la plasticidad celular y senescencia encondrocitos articulares: conexina 43 como diana terapéutica para el tratamiento de la artrosis / A novel therapeutic target for osteoarthritis: control of cellular plasticity and senescence using connexin43

Introducción: La artrosis (OA) es una enfermedad musculo esquelética degenerativa que afecta aproximadamente al13% de la población occidental. A día de hoy no existe un tratamiento eficaz que evite el progreso de la misma o facilite la regeneración del cartílago articular. La conexina43 (Cx43) es una proteína transmembrana que se encuentra en niveles elevados en el cartílago y en la membrana sinovial de pacientes con OA. Esta proteína forma canales que permiten el intercambio de moléculas e iones entre dos células en contacto o entre la célula y su entorno, denominados uniones co‐municantes (UCs) y hemicanales, respectivamente. En este estudio se investigó la función de la Cx43 y de las UCs en la degradación del cartílago articular de pacientes con OA. Material y métodos: Se han aislado condrocitos primarios del cartílago de donantes OA y sanos. Se evaluaron los niveles proteicos mediante Western blot, inmunofluorescencia y citometría de flujo. La expresión génica se ha evaluado mediante RT‐qPCR, mientras que la comunicación celular se estudió mediante el ensayo scrape loading/dye transfer. La senescencia celular se evaluó midiendo la actividad de la β‐galactosidasa mediante citometría celular o microscopía. Resultados: Los resultados obtenidos indican que la sobre actividad de la Cx43 y de la comunicación intercelular a través de UCs detectadas en OA están implicadas con el progreso de la enfermedad al activar procesos de desdiferenciación celular hacia un estado inmaduro y senescencia celular. Utilizando condrocitos en cultivo aislados del cartílago de donantes con OA hemos demostrado que el incremento de la Cx43 activa factores implicados en la transición epitelio‐me sénquima (TEM), como el factor de transcripción Twist‐1. El incremento en el número de células desdiferenciadas y con altos índices de proliferación celular desencadena en senescencia celular vía p53/p16INK4a, activando el fenotipo secretor asociado a senescencia (SASP, del inglés Senescence-Associated Secretory Phenotype) que incluye la síntesis y liberación de factores inflamatorios como la interleuquina 6 (IL‐6). La disminución de los niveles de la Cx43 utilizando pequeñas moléculas como la oleuropeína o técnicas de edición genética como CRISPR/Cas9 revirtió el proceso dando lugar a re‐diferenciación celular, mejorando el fenotipo celular con incremento en proteínas implicadas en formación del tejido y diminuyendo la síntesis de MMPs y del componente inflamatorio y senescencia. Conclusiones: La disminución de la Cx43 en condrocitos artrósicos restaura regeneración tisular, por activación de re‐diferenciación celular y disminución de senescencia. Estos resultados corroboran el uso de la Cx43 como una diana terapéutica eficaz para restaurar regeneración del cartílago en pacientes con OA y evitar la progresión de la enfermedad

Introduction: Osteoarthritis (OA) is a degenerative musculoskeletal disease, which affects approximately the 13% of western population. Nowadays, there is no effective treatment for OA to avoid disease progression or to promote cartilage regeneration. Connexin43 (Cx43) is a transmembrane protein increased in cartilage and synovium from OA patients.Cx43 forms membrane channels that allow the exchange of molecules and ions between two adjacent cells through gapjunctions (GJs), or between a cell and its environment through hemichannels. In this study we investigated the involvement of Cx43 and GJ intercellular communication in the degradation of articular cartilage in chondrocytes from patientswith OA. Material and methods:Primary chondrocytes were obtained from cartilage from OA and healthy donors. Protein levelswere evaluated by western‐blot, immunofluorescence and flow cytometry. RNA expression was evaluated by RT‐qPCR.A scrape loading/dye transfer assay was used to evaluate cell communication. Cell senescence was analysed by flowcytometry or by light microscopy using β‐galactosidase assay.Results:Cx43 and GJs overactivities were correlated with the progression of OA, by promoting chronic cell dedifferen‐tiation and senescence in vitroassays. We found that Cx43 over expression activates factors involved in epithelial‐to‐me‐senchymal transition, such as Twist‐1. Increased levels of dedifferentiated cells, with high rates of cell proliferation, led to cell senescence via p53/p16INK4a, activating the senescence‐associated secretory phenotype (SASP) and promoting the synthesis and liberation of inflammatory factors, including the interleukin‐6 (IL‐6). Cx43 down regulation by using small molecules, such as oleuropein, or by genetic edition with CRISPR technology, led to the chondrocyte redifferentiation and an improved phenotype, with increased synthesis of extracellular matrix proteins such as Col2A1 and down‐regulating the synthesis of MMPs, inflammation and senescence. Conclusions: Down regulation of Cx43 in OA chondrocytes restores regeneration by activating chondrocyte re‐differentiation and decreasing cellular senescence. These results corroborate the use of Cx43 as an effective therapeutic target in order to restore cartilage regeneration and avoid OA progression

All-trans retinoic acid restores gap junctional intercellular communication between oral cancer cells with upregulation of Cx32 and Cx43 expressions in vitro

Objective: All-trans retinoic acid (ATRA) has been demonstrated to inhibit tumor growth by restoration of gap junctional intercellular communication (GJIC) via upregulation of connexin (Cx) expression in some solid tumors. However, the relationship between ATRA and GJIC remains unclear in oral squamous cell carcinoma (OSCC). The aim of this study was to investigate the effect of ATRA on the GJIC function of OSCC. Study design: We measured the effects of ATRA on the viability and cell cycle distribution of SCC9 and Tca8113 OSCC cells. The GJIC function was observed using the scrape-loading dye transfer technique, and the mRNA and protein levels of Cx32 and Cx43 were detected by qRT-PCR, Western blot, and immunofluorescence assays. Results: ATRA inhibited the growth of OSCC cells in a dose- and time-dependent manner (P <0.05) and caused cell cycle arrest. ATRA-treated cells showed a 2.69-fold and 2.06-fold enhancement of GJIC in SCC9 and Tca8113 cells, respectively (P <0.05). Moreover, ATRA induced upregulation of Cx32 and Cx43 at both the mRNA and protein levels in OSCC cells. Conclusion: Our results indicated that restoration of GJIC via enhanced Cx32 and Cx43 expression might serve as a novel mechanism for the anti-tumor effect of ATRA in OSCC (AU)