Increased expression of the TRPM4 channel has been reported to be associated with the progression of prostate cancer. However, the molecular mechanism underlying its effect remains unknown. This work ...found that decreasing TRPM4 levels leads to the reduced proliferation of PC3 cells. This effect was associated with a decrease in total β‐catenin protein levels and its nuclear localization, and a significant reduction in Tcf/Lef transcriptional activity. Moreover, TRPM4 silencing increases the Ser33/Ser37/Thr41 β‐catenin phosphorylated population and reduces the phosphorylation of GSK‐3β at Ser9, suggesting an increase in β‐catenin degradation as the underlying mechanism. Conversely, TRPM4 overexpression in LNCaP cells increases the Ser9 inhibitory phosphorylation of GSK‐3β and the total levels of β‐catenin and its nonphosphorylated form. Finally, PC3 cells with reduced levels of TRPM4 showed a decrease in basal and stimulated phosphoactivation of Akt1, which is likely responsible for the decrease in GSK‐3β activity in these cells. Our results also suggest that the effect of TRPM4 on Akt1 is probably mediated by an alteration in the calcium/calmodulin‐EGFR axis, linking TRPM4 activity with the observed effects in β‐catenin‐related signaling pathways. These results suggest a role for TRPM4 channels in β‐catenin oncogene signaling and underlying mechanisms, highlighting this ion channel as a new potential target for future therapies in prostate cancer.
Here, we report that TRPM4 expression and its ion channel function positively regulate the β‐catenin stability, nuclear localization, and transcription co‐activator activity, enhancing the proliferation of prostate cancer cell lines, through a calcium/calmodulin‐EGFR axis, stimulating the Akt1 signaling and promoting the inhibitory phosphorylation of GSK‐3β. These findings highlight this ion channel as a new potential target for future therapies in prostate cancer.
Some chemoattractants and leukocytes such as M1 and M2 macrophages are known to be involved in the development of glomerulosclerosis during diabetic nephropathy (DN). In the course of diabetes, an ...altered and defective cellular metabolism leads to the increase in adenosine levels, and thus to changes in the polarity (M1/M2) of macrophages. MRS1754, a selective antagonist of the A2B adenosine receptor (A2BAR), attenuated glomerulosclerosis and decreased macrophage-myofibroblast transition in DN rats. Therefore, we aimed to investigate the effect of MRS1754 on the glomerular expression/secretion of chemoattractants, the intraglomerular infiltration of leukocytes, and macrophage polarity in DN rats. Kidneys/glomeruli of non-diabetic, DN, and MRS1754-treated DN rats were processed for transcriptomic analysis, immunohistopathology, ELISA, and in vitro macrophage migration assays. The transcriptomic analysis identified an upregulation of transcripts and pathways related to the immune system in the glomeruli of DN rats, which was attenuated using MRS1754. The antagonism of the A2BAR decreased glomerular expression/secretion of chemoattractants (CCL2, CCL3, CCL6, and CCL21), the infiltration of macrophages, and their polarization to M2 in DN rats. The in vitro macrophages migration induced by conditioned-medium of DN glomeruli was significantly decreased using neutralizing antibodies against CCL2, CCL3, and CCL21. We concluded that the pharmacological blockade of the A2BAR decreases the transcriptional expression of genes/pathways related to the immune response, protein expression/secretion of chemoattractants, as well as the infiltration of macrophages and their polarization toward the M2 phenotype in the glomeruli of DN rats, suggesting a new mechanism implicated in the antifibrotic effect of MRS1754.
Diabetic nephropathy (DN) is the main cause of end-stage renal disease, which remains incurable. The progression of DN is associated with progressive and irreversible renal fibrosis and also high ...levels of adenosine. Our aim was to evaluate the effects of ADORA3 antagonism on renal injury in streptozotocin-induced diabetic rats. An ADORA3 antagonist that was administered in diabetic rats greatly inhibited the levels of inflammatory interleukins IL-1β and IL-18, meanwhile when adenosine deaminase was administered, there was a non-selective attenuation of the inflammatory mediators IL-1β, IL-18, IL-6, and induction of IL-10. The ADORA3 antagonist attenuated the high glucose-induced activation of caspase 1 in HK2 cells in vitro. Additionally, ADORA3 antagonisms blocked the increase in caspase 1 and the nuclear localization of NFκB in the renal tubular epithelium of diabetic rats, both events that are involved in regulating the production and activation of IL-1β and IL-18. The effects of the A3 receptor antagonist resulted in the attenuation of kidney injury, as evidenced by decreased levels of the pro-fibrotic marker α-SMA at histological levels and the restoration of proteinuria in diabetic rats. We conclude that ADORA3 antagonism represents a potential therapeutic target that mechanistically works through the selective blockade of the NLRP3 inflammasome.
Diabetic nephropathy (DN) is considered the main cause of kidney disease in which myofibroblasts lead to renal fibrosis. Macrophages were recently identified as the major source of myofibroblasts in ...a process known as macrophage–myofibroblast transition (MMT). Adenosine levels increase during DN and in vivo administration of MRS1754, an antagonist of the A2B adenosine receptor (A2BAR), attenuated glomerular fibrosis (glomerulosclerosis). We aimed to investigate the association between A2BAR and MMT in glomerulosclerosis during DN. Kidneys/glomeruli of non-diabetic, diabetic, and MRS1754-treated diabetic (DM+MRS1754) rats were processed for histopathologic, transcriptomic, flow cytometry, and cellular in vitro analyses. Macrophages were used for in vitro cell migration/transmigration assays and MMT studies. In vivo MRS1754 treatment attenuated the clinical and histopathological signs of glomerulosclerosis in DN rats. Transcriptomic analysis demonstrated a decrease in chemokine-chemoattractants/cell-adhesion genes of monocytes/macrophages in DM+MRS1754 glomeruli. The number of intraglomerular infiltrated macrophages and MMT cells increased in diabetic rats. This was reverted by MRS1754 treatment. In vitro cell migration/transmigration decreased in macrophages treated with MRS1754. Human macrophages cultured with adenosine and/or TGF-β induced MMT, a process which was reduced by MRS1754. We concluded that pharmacologic blockade of A2BAR attenuated some clinical signs of renal dysfunction and glomerulosclerosis, and decreased intraglomerular macrophage infiltration and MMT in DN rats.
Deficient insulin signaling is a key event mediating diabetic glomerulopathy. Additionally, diabetic kidney disease has been related to increased levels of adenosine. Therefore, we tested a link ...between insulin deficiency and dysregulated activity of the equilibrative nucleoside transporters (ENTs) responsible for controlling extracellular levels of adenosine. In ex vivo glomeruli, high D-glucose decreased nucleoside uptake mediated by ENT1 and ENT2 transporters, resulting in augmented extracellular levels of adenosine. This condition was reversed by exposure to insulin. Particularly, insulin through insulin receptor/PI3K pathway markedly upregulated ENT2 uptake activity to restores the extracellular basal level of adenosine. Using primary cultured rat podocytes as a cellular model, we found insulin was able to increase ENT2 maximal velocity of transport. Also, PI3K activity was necessary to maintain ENT2 protein levels in the long term. In glomeruli of streptozotocin-induced diabetic rats, insulin deficiency leads to decreased activity of ENT2 and chronically increased extracellular levels of adenosine. Treatment of diabetic rats with adenosine deaminase attenuated both the glomerular loss of nephrin and proteinuria. In conclusion, we evidenced ENT2 as a target of insulin signaling and sensitive to dysregulation in diabetes, leading to chronically increased extracellular adenosine levels and thereby setting conditions conducive to kidney injury.
In type I diabetes mellitus (T1DM) pancreatic β cells are destroyed. Treatment entails exogenous insulin administration and strict diet control, yet optimal glycemic control is hardly attainable. ...Islet transplant could be an alternative in patients with poor glycemic control, but inefficient islet purification and autoimmune response of patients is still a challenge. For these reasons, it is necessary to explore new cellular sources and immunological isolation methods oriented to develop T1DM cell-based therapies.
We postulate human adipose-derived stem cell (hASC) as an adequate source to generate pancreatic islet cells in vitro, and to produce islet-like structures. Furthermore, we propose microencapsulation of these aggregates as an immunological isolation strategy.
hASC obtained from lipoaspirated fat tissue from human donors were differentiated in vitro to insulin (Ins) and glucagon (Gcg) producing cells. Then, insulin producing cells (IPC) and glucagon producing cells (GPC) were cocultured in low adhesion conditions to form cellular aggregates, and later encapsulated in a sodium alginate polymer. Expression of pancreatic lineage markers and secretion of insulin or glucagon in vitro were analyzed.
The results show that multipotent hASC efficiently differentiate to IPC and GPC, and express pancreatic markers, including insulin or glucagon hormones which they secrete upon stimulation (fivefold for insulin in IPC, and fourfold for glucagon, compared to undifferentiated cells). In turn, calculation of the Feret diameter and area of cellular aggregates revealed mean diameters of ~ 80 µm, and 65% of the aggregates reached 4000 µm
at 72 h of formation. IPC/GPC aggregates were then microencapsulated in sodium-alginate polymer microgels, which were found to be more stable when stabilized with Ba
, yielding average diameters of ~ 300 µm. Interestingly, Ba
-microencapsulated aggregates respond to high external glucose with insulin secretion.
The IPC/GPC differentiation process from hASC, followed by the generation of cellular aggregates that are later microencapsulated, could represent a possible treatment for T1DM.
Progressive diabetic nephropathy (DN) and loss of renal function correlate with kidney fibrosis. Crosstalk between TGF-β and adenosinergic signaling contributes to the phenotypic transition of cells ...and to renal fibrosis in DN models. We evaluated the role of TGF-β on NT5E gene expression coding for the ecto-5`-nucleotidase CD73, the limiting enzyme in extracellular adenosine production. We showed that high d-glucose may predispose HK-2 cells towards active transcription of the proximal promoter region of the NT5E gene while additional TGF-β results in full activation. The epigenetic landscape of the NT5E gene promoter was modified by concurrent TGF-β with occupancy by the p300 co-activator and the phosphorylated forms of the Smad2/3 complex and RNA Pol II. Transcriptional induction at NT5E in response to TGF-β was earlier compared to the classic responsiveness genes PAI-1 and Fn1. CD73 levels and AMPase activity were concomitantly increased by TGF-β in HK-2 cells. Interestingly, we found increased CD73 content in urinary extracellular vesicles only in diabetic patients with renal repercussions. Further, CD73-mediated AMPase activity was increased in the urinary sediment of DN patients. We conclude that the NT5E gene is a target of the profibrotic TGF-β cascade and is a traceable marker of progressive DN.
•The fibrogenic cascade of TGF-β/Smads targets the ecto-5′-nucleotidase gene.•TGF-β and high glucose synergistically modify the epigenetic landscape at this gene.•The gene product CD73 is a traceable marker of progressive diabetic nephropathy.
Several mechanisms directing a rapid transcriptional reactivation of genes immediately after mitosis have been described. However, little is known about the maintenance of repressive signals during ...mitosis. In this work, we address the role of Ski in the repression of gene expression during M/G1 transition in mouse embryonic fibroblasts (MEFs). We found that Ski localises as a distinct pair of dots at the pericentromeric region of mitotic chromosomes, and the absence of the protein is related to high acetylation and low tri-methylation of H3K9 in pericentromeric major satellite. Moreover, differential expression assays in early G1 cells showed that the presence of Ski is significantly associated with repression of genes localised nearby to pericentromeric DNA. In mitotic cells, chromatin immunoprecipitation assays confirmed the association of Ski to major satellite and the promoters of the most repressed genes: Mmp3, Mmp10 and Mmp13. These genes are at pericentromeric region of chromosome 9. In these promoters, the presence of Ski resulted in increased H3K9 tri-methylation levels. This Ski-dependent regulation is also observed during interphase. Consequently, Mmp activity is augmented in Ski−/− MEFs. Altogether, these data indicate that association of Ski with the pericentromeric region of chromosomes during mitosis is required to maintain the silencing bookmarks of underlying chromatin.
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•Here we show that Ski is associated with the pericentromeric region of mitotic chromosomes and it is required for H3K9me3 of major satellite.•In early G1, there is a Ski-dependent repression of genes located in or nearby pericentromeric region of chromosomes. The most repressed genes are Mmp3, Mmp10 and Mmp13.•During mitosis and interphase, Ski is associated with the promoter of these genes and is required for H3K9me3.•We propose a model where Ski remains associated with specific chromosomes at pericentromeric DNA in mitosis, driving methylation of H3K9 at major satellite and promoters of neighbouring genes, hence controlling heterochromatin structure, while preserving the repressive marks in genes, which must be kept silenced once the transcriptional program is resumed after cell division.
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