ERα36–GPER1 Collaboration Inhibits TLR4/NFκB-Induced Pro-Inflammatory Activity in Breast Cancer Cells

Inflammation is important for the initiation and progression of breast cancer. We have previously reported that in monocytes, estrogen regulates TLR4/NFκB-mediated inflammation via the interaction of the Erα isoform ERα36 with GPER1. We therefore investigated whether a similar mechanism is present in breast cancer epithelial cells, and the effect of ERα36 expression on the classic 66 kD ERα isoform (ERα66) functions. We report that estrogen inhibits LPS-induced NFκB activity and the expression of downstream molecules TNFα and IL-6. In the absence of ERα66, ERα36 and GPER1 are both indispensable for this effect. In the presence of ERα66, ERα36 or GPER1 knock-down partially inhibits NFκB-mediated inflammation. In both cases, ERα36 overexpression enhances the inhibitory effect of estrogen on inflammation. We also verify that ERα36 and GPER1 physically interact, especially after LPS treatment, and that GPER1 interacts directly with NFκB. When both ERα66 and ERα36 are expressed, the latter acts as an inhibitor of ERα66 via its binding to estrogen response elements. We also report that the activation of ERα36 leads to the inhibition of breast cancer cell proliferation. Our data support that ERα36 is an inhibitory estrogen receptor that, in collaboration with GPER1, inhibits NFκB-mediated inflammation and ERα66 actions in breast cancer cells.     

HPTE-Induced Embryonic Thymocyte Death and Alteration of Differentiation Is Not Rescued by ERα or GPER Inhibition but Is Exacerbated by Concurrent TCR Signaling

Organochlorine pesticides, such as DDT, methoxychlor, and their metabolites, have been characterized as endocrine disrupting chemicals (EDCs); suggesting that their modes of action involve interaction with or abrogation of endogenous endocrine function. This study examined whether embryonic thymocyte death and alteration of differentiation induced by the primary metabolite of methoxychlor, HPTE, rely upon estrogen receptor binding and concurrent T cell receptor signaling. Estrogen receptor inhibition of ERα or GPER did not rescue embryonic thymocyte death induced by HPTE or the model estrogen diethylstilbestrol (DES). Moreover, adverse effects induced by HPTE or DES were worsened by concurrent TCR and CD2 differentiation signaling, compared with EDC exposure post-signaling. Together, these data suggest that HPTE- and DES-induced adverse effects on embryonic thymocytes do not rely solely on ER alpha or GPER but may require both. These results also provide evidence of a potential collaborative signaling mechanism between TCR and estrogen receptors to mediate adverse effects on embryonic thymocytes, as well as highlight a window of sensitivity that modulates EDC exposure severity.     

Functional Significance of Selective Expression of ERα and ERβ in Mammary Gland Organ Culture

Thoracic pair of mammary glands from steroid hormone-pretreated mice respond to hormones structurally and functionally in organ culture. A short exposure of glands for 24 h to 7,12 Dimethylbenz(a)anthracene (DMBA) during a 24-day culture period induced alveolar or ductal lesions. Methods: To differentiate the functional significance of ERα and ERβ, we employed estrogen receptor (ER) knockout mice. We compared the effects of DMBA on the development of preneoplastic lesions in the glands in the absence of ERα (αERKO) and ERβ (βERKO) using an MMOC protocol. Glands were also subjected to microarray analyses. We showed that estradiol can be replaced by EGF for pretreatment of mice. The carcinogen-induced lesions developed under both steroids and EGF pretreatment protocols. The glands from αERKO did not develop any lesions, whereas in βERKO mice in which ERα is intact, mammary alveolar lesions developed. Comparison of microarrays of control, αERKO and βERKO mice showed that ERα was largely responsible for proliferation and the MAP kinase pathways, whereas ERβ regulated steroid metabolism-related genes. The results indicate that ERα is essential for the development of precancerous lesions. Both subtypes, ERα and Erβ, differentially regulated gene expression in mammary glands in organ cultures.     

Extracellular Acidosis Differentially Regulates Estrogen Receptor β-Dependent EMT Reprogramming in Female and Male Melanoma Cells

Clinical outcomes of melanoma patients pointed out a gender disparity that supports a correlation between sex hormone activity on estrogen receptors (ER) and melanoma development and progression. Here, we found that the epithelial-to-mesenchymal transition (EMT) of melanoma cells induced by extracellular acidosis, which is a crucial hallmark of solid cancers, correlates with the expression of ERβ, the most representative ER on melanoma cells. Extracellular acidosis induces an enhanced expression of ERβ in female cells and EMT markers remain unchanged, while extracellular acidosis did not induce the expression of ERβ in male cells and EMT was strongly promoted. An inverse relationship between ERβ expression and EMT markers in melanoma cells of different sex exposed to extracellular acidosis was revealed by two different technical approaches: florescence-activated cell sorting of high ERβ expressing cell subpopulations and ERβ receptor silencing. Finally, we found that ERβ regulates EMT through NF-κB activation. These results demonstrate that extracellular acidosis drives a differential ERβ regulation in male and female melanoma cells and that this gender disparity might open new perspectives for personalized therapeutic approaches.     

Kisspeptin Neurons and Estrogen–Estrogen Receptor α Signaling: Unraveling the Mystery of Steroid Feedback System Regulating Mammalian Reproduction

Estrogen produced by ovarian follicles plays a key role in the central mechanisms controlling reproduction via regulation of gonadotropin-releasing hormone (GnRH) release by its negative and positive feedback actions in female mammals. It has been well accepted that estrogen receptor α (ERα) mediates both estrogen feedback actions, but precise targets had remained as a mystery for decades. Ever since the discovery of kisspeptin neurons as afferent ERα-expressing neurons to govern GnRH neurons, the mechanisms mediating estrogen feedback are gradually being unraveled. The present article overviews the role of kisspeptin neurons in the arcuate nucleus (ARC), which are considered to drive pulsatile GnRH/gonadotropin release and folliculogenesis, in mediating the estrogen negative feedback action, and the role of kisspeptin neurons located in the anteroventral periventricular nucleus-periventricular nucleus (AVPV-PeN), which are thought to drive GnRH/luteinizing hormone (LH) surge and consequent ovulation, in mediating the estrogen positive feedback action. This implication has been confirmed by the studies showing that estrogen-bound ERα down- and up-regulates kisspeptin gene (Kiss1) expression in the ARC and AVPV-PeN kisspeptin neurons, respectively. The article also provides the molecular and epigenetic mechanisms regulating Kiss1 expression in kisspeptin neurons by estrogen. Further, afferent ERα-expressing neurons that may regulate kisspeptin release are discussed.     

Screening of Relevant Metabolism-Disrupting Chemicals on Pancreatic β-Cells: Evaluation of Murine and Human In Vitro Models

Endocrine-disrupting chemicals (EDCs) are chemical substances that can interfere with the normal function of the endocrine system. EDCs are ubiquitous and can be found in a variety of consumer products such as food packaging materials, personal care and household products, plastic additives, and flame retardants. Over the last decade, the impact of EDCs on human health has been widely acknowledged as they have been associated with different endocrine diseases. Among them, a subset called metabolism-disrupting chemicals (MDCs) is able to promote metabolic changes that can lead to the development of metabolic disorders such as diabetes, obesity, hepatic steatosis, and metabolic syndrome, among others. Despite this, today, there are still no definitive and standardized in vitro tools to support the metabolic risk assessment of existing and emerging MDCs for regulatory purposes. Here, we evaluated the following two different pancreatic cell-based in vitro systems: the murine pancreatic β-cell line MIN6 as well as the human pancreatic β-cell line EndoC-βH1. Both were challenged with the following range of relevant concentrations of seven well-known EDCs: (bisphenol-A (BPA), bisphenol-S (BPS), bisphenol-F (BPF), perfluorooctanesulfonic acid (PFOS), di(2-ethylhexyl) phthalate (DEHP), cadmium chloride (CdCl₂), and dichlorodiphenyldichloroethylene (DDE)). The screening revealed that most of the tested chemicals have detectable, deleterious effects on glucose-stimulated insulin release, insulin content, electrical activity, gene expression, and/or viability. Our data provide new molecular information on the direct effects of the selected chemicals on key aspects of pancreatic β-cell function, such as the stimulus-secretion coupling and ion channel activity. In addition, we found that, in general, the sensitivity and responses were comparable to those from other in vivo studies reported in the literature. Overall, our results suggest that both systems can serve as effective tools for the rapid screening of potential MDC effects on pancreatic β-cell physiology as well as for deciphering and better understanding the molecular mechanisms that underlie their action.     

In Vitro Assays to Identify Metabolism-Disrupting Chemicals with Diabetogenic Activity in a Human Pancreatic β-Cell Model

There is a need to develop identification tests for Metabolism Disrupting Chemicals (MDCs) with diabetogenic activity. Here we used the human EndoC-βH1 β-cell line, the rat β-cell line INS-1E and dispersed mouse islet cells to assess the effects of endocrine disruptors on cell viability and glucose-stimulated insulin secretion (GSIS). We tested six chemicals at concentrations within human exposure (from 0.1 pM to 1 µM). Bisphenol-A (BPA) and tributyltin (TBT) were used as controls while four other chemicals, namely perfluorooctanoic acid (PFOA), triphenylphosphate (TPP), triclosan (TCS) and dichlorodiphenyldichloroethylene (DDE), were used as “unknowns”. Regarding cell viability, BPA and TBT increased cell death as previously observed. Their mode of action involved the activation of estrogen receptors and PPARγ, respectively. ROS production was a consistent key event in BPA-and TBT-treated cells. None of the other MDCs tested modified viability or ROS production. Concerning GSIS, TBT increased insulin secretion while BPA produced no effects. PFOA decreased GSIS, suggesting that this chemical could be a “new” diabetogenic agent. Our results indicate that the EndoC-βH1 cell line is a suitable human β-cell model for testing diabetogenic MDCs. Optimization of the test methods proposed here could be incorporated into a set of protocols for the identification of MDCs.     

New Therapeutic Targets for Hepatic Fibrosis in the Integrin Family, α8β1 and α11β1, Induced Specifically on Activated Stellate Cells

A huge effort has been devoted to developing drugs targeting integrins over 30 years, because of the primary roles of integrins in the cell-matrix milieu. Five αv-containing integrins, in the 24 family members, have been a central target of fibrosis. Currently, a small molecule against αvβ1 is undergoing a clinical trial for NASH-associated fibrosis as a rare agent aiming at fibrogenesis. Latent TGFβ activation, a distinct talent of αv-integrins, has been intriguing as a therapeutic target. None of the αv-integrin inhibitors, however, has been in the clinical market. αv-integrins commonly recognize an Arg-Gly-Asp (RGD) sequence, and thus the pharmacophore of inhibitors for the 5-integrins is based on the same RGD structure. The RGD preference of the integrins, at the same time, dilutes ligand specificity, as the 5-integrins share ligands containing RGD sequence such as fibronectin. With the inherent little specificity in both drugs and targets, “disease specificity” has become less important for the inhibitors than blocking as many αv-integrins. In fact, an almighty inhibitor for αv-integrins, pan-αv, was in a clinical trial. On the contrary, approved integrin inhibitors are all specific to target integrins, which are expressed in a cell-type specific manner: αIIbβ3 on platelets, α4β1, α4β7 and αLβ2 on leukocytes. Herein, “disease specific” integrins would serve as attractive targets. α8β1 and α11β1 are selectively expressed in hepatic stellate cells (HSCs) and distinctively induced upon culture activation. The exceptional specificity to activated HSCs reflects a rather “pathology specific” nature of these new integrins. The monoclonal antibodies against α8β1 and α11β1 in preclinical examinations may illuminate the road to the first medical agents.     

Bone Mass and Osteoblast Activity Are Sex-Dependent in Mice Lacking the Estrogen Receptor α in Chondrocytes and Osteoblast Progenitor Cells

While estrogen receptor alpha (ERα) is known to be important for bone development and homeostasis, its exact function during osteoblast differentiation remains unclear. Conditional deletion of ERα during specific stages of osteoblast differentiation revealed different bone phenotypes, which were also shown to be sex-dependent. Since hypertrophic chondrocytes can transdifferentiate into osteoblasts and substantially contribute to long-bone development, we aimed to investigate the effects of ERα deletion in both osteoblast and chondrocytes on bone development and structure. Therefore, we generated mice in which the ERα gene was inactivated via a Runx2-driven cyclic recombinase (ERα^{fl/fl; Runx2Cre}). We analyzed the bones of 3-month-old ERα^{fl/fl; Runx2Cre} mice by biomechanical testing, micro-computed tomography, and cellular parameters by histology. Male ERα^{fl/fl; Runx2Cre} mice displayed a significantly increased cortical bone mass and flexural rigidity of the femurs compared to age-matched controls with no active Cre-transgene (ERα^fl/fl). By contrast, female ERα^{fl/fl; Runx2Cre} mice exhibited significant trabecular bone loss, whereas in cortical bone periosteal and endosteal diameters were reduced. Our results indicate that the ERα in osteoblast progenitors and hypertrophic chondrocytes differentially contributes to bone mass regulation in male and female mice and improves our understanding of ERα signaling in bone cells in vivo.     

Early Inactivation of Membrane Estrogen Receptor Alpha (ERα) Recapitulates the Endothelial Dysfunction of Aged Mouse Resistance Arteries

Flow-mediated dilation (FMD) of resistance arteries is essential for tissue perfusion but it decreases with ageing. As estrogen receptor alpha (Erα encoded by Esr1), and more precisely membrane ERα, plays an important role in FMD in young mice in a ligand-independent fashion, we evaluated its influence on this arteriolar function in ageing. We first confirmed that in young (6-month-old) mice, FMD of mesenteric resistance arteries was reduced in Esr1−/− (lacking ERα) and C451A-ERα (lacking membrane ERα). In old (24-month-old) mice, FMD was reduced in WT mice compared to young mice, whereas it was not further decreased in Esr1−/− and C451A-ERα mice. Markers of oxidative stress were similarly increased in old WT and C451A-ERα mice. Reduction in oxidative stress with superoxide dismutase plus catalase or Mito-tempo, which reduces mitochondrial superoxide restored FMD to a normal control level in young C451A-ERα mice as well as in old WT mice and old C451A-ERα mice. Estradiol-mediated dilation was absent in old WT mice. We conclude that oxidative stress is a key event in the decline of FMD, and that an early defect in membrane ERα recapitulates phenotypically and functionally ageing of these resistance arteries. The loss of this function could take part in vascular ageing.     

A Regulatory Axis between Epithelial Splicing Regulatory Proteins and Estrogen Receptor α Modulates the Alternative Transcriptome of Luminal Breast Cancer

Epithelial splicing regulatory proteins 1 and 2 (ESRP1/2) control the splicing pattern during epithelial to mesenchymal transition (EMT) in a physiological context and in cancer, including breast cancer (BC). Here, we report that ESRP1, but not ESRP2, is overexpressed in luminal BCs of patients with poor prognosis and correlates with estrogen receptor α (ERα) levels. Analysis of ERα genome-binding profiles in cell lines and primary breast tumors showed its binding in the proximity of ESRP1 and ESRP2 genes, whose expression is strongly decreased by ERα silencing in hormone-deprived conditions. The combined knock-down of ESRP1/2 in MCF-7 cells followed by RNA-Seq, revealed the dysregulation of 754 genes, with a widespread alteration of alternative splicing events (ASEs) of genes involved in cell signaling, metabolism, cell growth, and EMT. Functional network analysis of ASEs correlated with ESRP1/2 expression in ERα+ BCs showed RAC1 as the hub node in the protein–protein interactions altered by ESRP1/2 silencing. The comparison of ERα- and ESRP-modulated ASEs revealed 63 commonly regulated events, including 27 detected in primary BCs and endocrine-resistant cell lines. Our data support a functional implication of the ERα-ESRP1/2 axis in the onset and progression of BC by controlling the splicing patterns of related genes.     

Recombinant Integrin β1 Signal Peptide Blocks Gliosis Induced by Aβ Oligomers

Glial cells participate actively in the early cognitive decline in Alzheimer’s disease (AD) pathology. In fact, recent studies have found molecular and functional abnormalities in astrocytes and microglia in both animal models and brains of patients suffering from this pathology. In this regard, reactive gliosis intimately associated with amyloid plaques has become a pathological hallmark of AD. A recent study from our laboratory reports that astrocyte reactivity is caused by a direct interaction between amyloid beta (Aβ) oligomers and integrin β1. Here, we have generated four recombinant peptides including the extracellular domain of integrin β1, and evaluated their capacity both to bind in vitro to Aβ oligomers and to prevent in vivo Aβ oligomer-induced gliosis and endoplasmic reticulum stress. We have identified the minimal region of integrin β1 that binds to Aβ oligomers. This region is called signal peptide and corresponds to the first 20 amino acids of the integrin β1 N-terminal domain. This recombinant integrin β1 signal peptide prevented Aβ oligomer-induced ROS generation in primary astrocyte cultures. Furthermore, we carried out intrahippocampal injection in adult mice of recombinant integrin β1 signal peptide combined with or without Aβ oligomers and we evaluated by immunohistochemistry both astrogliosis and microgliosis as well as endoplasmic reticulum stress. The results show that recombinant integrin β1 signal peptide precluded both astrogliosis and microgliosis and endoplasmic reticulum stress mediated by Aβ oligomers in vivo. We have developed a molecular tool that blocks the activation of the molecular cascade that mediates gliosis via Aβ oligomer/integrin β1 signaling.     

A Role for TGFβ Signaling in Preclinical Osteolytic Estrogen Receptor-Positive Breast Cancer Bone Metastases Progression

While tumoral Smad-mediated transforming growth factor β (TGFβ) signaling drives osteolytic estrogen receptor α-negative (ER-) breast cancer bone metastases (BMETs) in preclinical models, its role in ER+ BMETs, representing the majority of clinical BMETs, has not been documented. Experiments were undertaken to examine Smad-mediated TGFβ signaling in human ER+ cells and bone-tropic behavior following intracardiac inoculation of estrogen (E₂)-supplemented female nude mice. While all ER+ tumor cells tested (ZR-75-1, T47D, and MCF-7-derived) expressed TGFβ receptors II and I, only cells with TGFβ-inducible Smad signaling (MCF-7) formed osteolytic BMETs in vivo. Regulated secretion of PTHrP, an osteolytic factor expressed in >90% of clinical BMETs, also tracked with osteolytic potential; TGFβ and E₂ each induced PTHrP in bone-tropic or BMET-derived MCF-7 cells, with the combination yielding additive effects, while in cells not forming BMETs, PTHrP was not induced. In vivo treatment with 1D11, a pan-TGFβ neutralizing antibody, significantly decreased osteolytic ER+ BMETs in association with a decrease in bone-resorbing osteoclasts at the tumor-bone interface. Thus, TGFβ may also be a driver of ER+ BMET osteolysis. Moreover, additive pro-osteolytic effects of tumoral E₂ and TGFβ signaling could at least partially explain the greater propensity for ER+ tumors to form BMETs, which are primarily osteolytic.     

TNF-α and IL-1β Modulate Blood-Brain Barrier Permeability and Decrease Amyloid-β Peptide Efflux in a Human Blood-Brain Barrier Model

The blood-brain barrier (BBB) is a selective barrier and a functional gatekeeper for the central nervous system (CNS), essential for maintaining brain homeostasis. The BBB is composed of specialized brain endothelial cells (BECs) lining the brain capillaries. The tight junctions formed by BECs regulate paracellular transport, whereas transcellular transport is regulated by specialized transporters, pumps and receptors. Cytokine-induced neuroinflammation, such as the tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), appear to play a role in BBB dysfunction and contribute to the progression of Alzheimer’s disease (AD) by contributing to amyloid-β (Aβ) peptide accumulation. Here, we investigated whether TNF-α and IL-1β modulate the permeability of the BBB and alter Aβ peptide transport across BECs. We used a human BBB in vitro model based on the use of brain-like endothelial cells (BLECs) obtained from endothelial cells derived from CD34+ stem cells cocultivated with brain pericytes. We demonstrated that TNF-α and IL-1β differentially induced changes in BLECs’ permeability by inducing alterations in the organization of junctional complexes as well as in transcelluar trafficking. Further, TNF-α and IL-1β act directly on BLECs by decreasing LRP1 and BCRP protein expression as well as the specific efflux of Aβ peptide. These results provide mechanisms by which CNS inflammation might modulate BBB permeability and promote Aβ peptide accumulation. A future therapeutic intervention targeting vascular inflammation at the BBB may have the therapeutic potential to slow down the progression of AD.     

Opposing Roles of GSK3α and GSK3β Phosphorylation in Platelet Function and Thrombosis

One of the mechanisms by which PI3 kinase can regulate platelet function is through phosphorylation of downstream substrates, including glycogen synthase kinase-3 (GSK3)α and GSK3β. Platelet activation results in the phosphorylation of an N-terminal serine residue in GSK3α (Ser21) and GSK3β (Ser9), which competitively inhibits substrate phosphorylation. However, the role of phosphorylation of these paralogs is still largely unknown. Here, we employed GSK3α/β phosphorylation-resistant mouse models to explore the role of this inhibitory phosphorylation in regulating platelet activation. Expression of phosphorylation-resistant GSK3α/β reduced thrombin-mediated platelet aggregation, integrin α_IIbβ₃ activation, and α-granule secretion, whereas platelet responses to the GPVI agonist collagen-related peptide (CRP-XL) were significantly enhanced. GSK3 single knock-in lines revealed that this divergence is due to differential roles of GSK3α and GSK3β phosphorylation in regulating platelet function. Expression of phosphorylation-resistant GSK3α resulted in enhanced GPVI-mediated platelet activation, whereas expression of phosphorylation-resistant GSK3β resulted in a reduction in PAR-mediated platelet activation and impaired in vitro thrombus formation under flow. Interestingly, the latter was normalised in double GSK3α/β KI mice, indicating that GSK3α KI can compensate for the impairment in thrombosis caused by GSK3β KI. In conclusion, our data indicate that GSK3α and GSK3β have differential roles in regulating platelet function.     

Biphasic α2β1 Integrin Expression in Breast Cancer Metastasis to Bone

Integrins participate in the pathogenesis and progression of tumors at many stages during the metastatic cascade. However, current evidence for the role of integrins in breast cancer progression is contradictory and seems to be dependent on tumor stage, differentiation status, and microenvironmental influences. While some studies suggest that loss of α2β1 enhances cancer metastasis, other studies suggest that this integrin is pro-tumorigenic. However, few studies have looked at α2β1 in the context of bone metastasis. In this study, we aimed to understand the role of α2β1 integrin in breast cancer metastasis to bone. To address this, we utilized in vivo models of breast cancer metastasis to bone using MDA-MB-231 cells transfected with an α2 expression plasmid (MDA-OEα2). MDA cells overexpressing the α2 integrin subunit had increased primary tumor growth and dissemination to bone but had no change in tumor establishment and bone destruction. Further in vitro analysis revealed that tumors in the bone have decreased α2β1 expression and increased osteolytic signaling compared to primary tumors. Taken together, these data suggest an inverse correlation between α2β1 expression and bone-metastatic potential. Inhibiting α2β1 expression may be beneficial to limit the expansion of primary tumors but could be harmful once tumors have established in bone.     

Collagen I Modifies Connexin-43 Hemichannel Activity via Integrin α2β1 Binding in TGFβ1-Evoked Renal Tubular Epithelial Cells

Chronic Kidney Disease (CKD) is associated with sustained inflammation and progressive fibrosis, changes that have been linked to altered connexin hemichannel-mediated release of adenosine triphosphate (ATP). Kidney fibrosis develops in response to increased deposition of extracellular matrix (ECM), and up-regulation of collagen I is an early marker of renal disease. With ECM remodeling known to promote a loss of epithelial stability, in the current study we used a clonal human kidney (HK2) model of proximal tubular epithelial cells to determine if collagen I modulates changes in cell function, via connexin-43 (Cx43) hemichannel ATP release. HK2 cells were cultured on collagen I and treated with the beta 1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFβ1) ± the Cx43 mimetic Peptide 5 and/or an anti-integrin α2β1 neutralizing antibody. Phase microscopy and immunocytochemistry observed changes in cell morphology and cytoskeletal reorganization, whilst immunoblotting and ELISA identified changes in protein expression and secretion. Carboxyfluorescein dye uptake and biosensing measured hemichannel activity and ATP release. A Cytoselect extracellular matrix adhesion assay assessed changes in cell-substrate interactions. Collagen I and TGFβ1 synergistically evoked increased hemichannel activity and ATP release. This was paralleled by changes to markers of tubular injury, partly mediated by integrin α2β1/integrin-like kinase signaling. The co-incubation of the hemichannel blocker Peptide 5, reduced collagen I/TGFβ1 induced alterations and inhibited a positive feedforward loop between Cx43/ATP release/collagen I. This study highlights a role for collagen I in regulating connexin-mediated hemichannel activity through integrin α2β1 signaling, ahead of establishing Peptide 5 as a potential intervention.