Gastric cancer (GC) is a major health concern worldwide, presenting a complex pathophysiology that has hindered many therapeutic efforts so far. In this context, purinergic signaling emerges as a promising pathway for intervention due to its known role in cancer cell proliferation and migration. In this work, we explored in more detail the role of purinergic signaling in GC with several experimental approaches. First, we measured extracellular ATP concentrations on GC-derived cell lines (AGS, MKN-45, and MKN-74), finding higher levels of extracellular ATP than those obtained for the non-tumoral gastric cell line GES-1. Next, we established the P2Y2 and P2X4 receptors (P2Y2R and P2X4R) expression profile on these cells and evaluated their role on cell proliferation and migration after applying overexpression and knockdown strategies. In general, a P2Y2R overexpression and P2X4R downregulation pattern were observed on GC cell lines, and when these patterns were modified, concomitant changes in cell viability were observed. These modifications on gene expression also modified transepithelial electrical resistance (TEER), showing that higher P2Y2R levels decreased TEER, and high P2X4R expression had the opposite effect, suggesting that P2Y2R and P2X4R activation could promote and suppress epithelial-mesenchymal transition (EMT), respectively. These effects were confirmed after treating AGS cells with UTP, a P2Y2R-agonist that modified the expression patterns towards mesenchymal markers. To further characterize the effects of P2Y2R activation on EMT, we used cDNA microarrays and observed that UTP induced important transcriptional changes on several cell processes like cell proliferation induction, apoptosis inhibition, cell differentiation induction, and cell adhesion reduction. These results suggest that purinergic signaling plays a complex role in GC pathophysiology, and changes in purinergic balance can trigger tumorigenesis in non-tumoral gastric cells.

Inflammatory and wound healing responses take place during liver damage, primarily in the parenchymal tissue. It is known that cellular injury elicits an activation of the purinergic signaling, mainly by the P2X7 receptor; however, the role of P2Y receptors in the onset of liver pathology such as fibrosis has not been explored. Hence, we used mice treated with the hepatotoxin CCl₄ to implement a reversible model of liver fibrosis to evaluate the expression and function of the P2Y2 receptor (P2Y2R). Fibrotic livers showed an enhanced expression of P2Y2R that eliminated its zonal distribution. Hepatocytes from CCl₄-treated mice showed an exacerbated ERK-phosphorylated response to the P2Y2R-specific agonist, UTP. Cell proliferation was also enhanced in the fibrotic livers. Hepatic transcriptional analysis by microarrays, upon CCl₄ administration, showed that P2Y2 activation regulated diverse pathways, revealing complex action mechanisms. In conclusion, our data indicate that P2Y2R activation is involved in the onset of the fibrotic damage associated with the reversible phase of the hepatic damage promoted by CCl₄.

BACKGROUND: Vascular calcification is a major contributor to cardiovascular morbidity and mortality in diabetes and is driven in part by osteogenic reprogramming of vascular smooth muscle cells (SMCs). Diabetes is also associated with circadian rhythm disruption; however, how circadian regulators contribute to vascular calcification remains poorly understood. We investigated the role of the core circadian protein Bmal1 (basic helix-loop-helix ARNT-like protein 1) in diabetes-associated vascular calcification. METHODS: Bmal1 expression was examined in diabetic mouse aortas, human diabetic arterial tissues, and vascular SMCs exposed to high glucose. SMC-specific Bmal1 knockout mice were generated and subjected to low-dose streptozotocin-induced diabetes. Vascular calcification and stiffness were assessed by calcium staining and quantification, and pulse wave velocity. RNA sequencing, chromatin immunoprecipitation, luciferase reporter assay, and clustered regularly interspaced short palindromic repeats-clustered regularly interspaced short palindromic repeat–associated 9–mediated genome editing were used to define Bmal1-regulated molecular mechanisms. RESULTS: Bmal1, but not other circadian regulators, was selectively upregulated in diabetic arteries and vascular SMCs under hyperglycemic conditions. SMC-specific deletion of Bmal1 markedly attenuated diabetes-induced vascular calcification, reduced aortic stiffness, and suppressed osteogenic gene expression in vivo and in vitro. RNA-sequencing analysis revealed enrichment of Bmal1-dependent genes involved in osteogenic differentiation and extracellular matrix organization. Mechanistically, Bmal1 directly binds to an E-box motif in the Runx2 (Runt-related transcription factor 2) promoter, enhancing Runx2 transcription independently of canonical circadian pathways. Disruption of Bmal1 binding to the Runx2 promoter abolished Bmal1-induced Runx2 expression. CONCLUSIONS: Our findings identify Bmal1 as a key mediator of diabetes-induced vascular calcification through direct transcriptional regulation of Runx2 via a noncanonical circadian mechanism. These results reveal a previously unrecognized pathogenic role for Bmal1 in vascular disease and suggest that targeting dysregulated circadian factors may represent a novel therapeutic strategy for preventing vascular calcification in diabetes.

Purinergic signaling is a sophisticated system of elements in which ATP and related molecules function as intercellular messengers. When ATP is released into the extracellular space, it activates specific receptors that belong to the P2 family. In parallel, ectonucleotidases transform ATP in its dephosphorylated metabolites including adenosine, which stimulates P1 receptors. The activity of both receptors influences various cellular processes. Moreover, metabolic conditions are concatenated with purine signaling to conform a dynamic and continuous informational network. The role of purinergic signaling in ovarian cells has been investigated, for instance, it is known that cells conforming the follicle express functional receptors that modulate basic cellular process such as proliferation, induction of apoptotic cell death, and steroidogenesis. In this chapter, we review contemporary information on purinergic action in ovarian cell physiology and state its relevance in this field.