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Sig1R silencing inhibits CRC HCT-116 cell motility

The sigma 1 receptor (Sig1R) is a stress-activated chaperone that regulates ion channels and is associated with pathologic conditions, such as stroke, neurodegenerative diseases, and addiction. Aberrant expression levels of ion channels and Sig1R have been detected in tumors and cancer cells, such as myeloid leukemia and colorectal cancer, but the link between ion channel regulation and Sig1R overexpression during malignancy has not been established. In this study, we found that Sig1R dynamically controls the membrane expression of the human voltage-dependent K+ channel human ether-à-go-go-related gene (hERG) in myeloid leukemia and colorectal cancer cell lines. Sig1R promoted the formation of hERG/β1-integrin signaling complexes upon extracellular matrix stimulation, triggering the activation of the PI3K/AKT pathway. Consequently, the presence of Sig1R in cancer cells increased motility and VEGF secretion. In vivo, Sig1R expression enhanced the aggressiveness of tumor cells by potentiating invasion and angiogenesis, leading to poor survival. Collectively, our findings highlight a novel function for Sig1R in mediating cross-talk between cancer cells and their microenvironment, thus driving oncogenesis by shaping cellular electrical activity in response to extracellular signals. Given the involvement of ion channels in promoting several hallmarks of cancer, our study also offers a potential strategy to therapeutically target ion channel function through Sig1R inhibition. Cancer Res; 76(3); 607–18. ©2015 AACR.

Sig1R is associated to β1 integrin and promotes β1 integrin / hERG association without altering β1 integrin membrane expression

Control experiments showing specificity of PLA assay between hERG and β1 integrin

Sig1R promotes hERG- and AKT-dependent FDM-induced cell spreading, VEGF secretion and endothelial transmigration

FDM induces a stable increase in hERG current in a Sig1R-dependent manner but does not alter channel maturation

A Correction to this paper has been published: https://doi.org/10.1038/s42003-020-01502-2.

The sigma 1 receptor (Sig1R) is a stress-activated chaperone that regulates ion channels and is associated with pathologic conditions, such as stroke, neurodegenerative diseases, and addiction. Aberrant expression levels of ion channels and Sig1R have been detected in tumors and cancer cells, such as myeloid leukemia and colorectal cancer, but the link between ion channel regulation and Sig1R overexpression during malignancy has not been established. In this study, we found that Sig1R dynamically controls the membrane expression of the human voltage-dependent K+ channel human ether-à-go-go-related gene (hERG) in myeloid leukemia and colorectal cancer cell lines. Sig1R promoted the formation of hERG/β1-integrin signaling complexes upon extracellular matrix stimulation, triggering the activation of the PI3K/AKT pathway. Consequently, the presence of Sig1R in cancer cells increased motility and VEGF secretion. In vivo, Sig1R expression enhanced the aggressiveness of tumor cells by potentiating invasion and angiogenesis, leading to poor survival. Collectively, our findings highlight a novel function for Sig1R in mediating cross-talk between cancer cells and their microenvironment, thus driving oncogenesis by shaping cellular electrical activity in response to extracellular signals. Given the involvement of ion channels in promoting several hallmarks of cancer, our study also offers a potential strategy to therapeutically target ion channel function through Sig1R inhibition. Cancer Res; 76(3); 607–18. ©2015 AACR.

Active urea transport in the rat inner medullary collecting duct: Functional characterization and initial expression cloning. Active transport of urea has been proposed to exist inthe inner medullary collecting duct (IMCD) of low-protein fed mammals for over 30 years. We perfused IMCD subsegments from rats fed a standard (18%) or a low (8%) protein diet and tested for the presence of active urea transport. We found no active urea transport in terminal IMCDs, regardless of diet. In initial IMCDs from rats fed 18% protein or fed 8% protein for one to two weeks, we again found no active urea transport. However, in rats fed 8% protein for three to four weeks, we found significant net urea reabsorption. This active urea reabsorption was inhibited when Na + ,K + -ATPase activity was inhibited by adding 1mM ouabain or removing bath potassium, suggesting a secondary active transport process. Removing sodium from the perfusate completely inhibited net urea reabsorption, demonstrating that this active urea transport is dependent upon the presence of sodium in the tubule lumen. Unlike the facilitated urea transporter, the active urea transporter was not inhibited by phloretin nor stimulated by vasopressin, suggesting that it is a distinct transport protein. To test this hypothesis, we size-separated poly(A) + -RNA preparedfrom inner medullae of rats fed 8% protein for three weeks and injected it into Xenopus laevis oocytes. RNA froma 4.4 to 8.4kb size fraction increased urea permeability fourfold compared to water-injected oocytes or injecting RNA from other size-fractions. We conclude that feeding rats a low-protein diet for three weeks induces the expression of an unique, secondary active, sodium-dependent urea transporter whose cDNA is between 4.4 and 8.4kb in size. In addition, our results suggest that it will be possible to clone the cDNA for this sodium-urea cotransporter by expression in Xenopus laevis oocytes.

International audience; In mammals, urea is the predominant end-product of nitrogen metabolism and plays a central role in the urinary-concentrating mechanism. Urea accumulation in the renal medulla is critical to the ability of the kidney to concentrate urine to an osmolality greater than systemic plasma. Regulation of urea excretion and accumulation in the renal medulla depends on the functional state of specialized phloretin-sensitive urea transporters. To study these transporters and their regulation of expression we isolated a cDNA which encodes the rat homologue (rUT2) of rabbit UT2 (You, G., C. P. Smith, Y. Kanai, W.-S. Lee, M. Stelzner, and M. A. Hediger, et al. Nature (Lond.). 1993.365:844-847). Rat UT2 has 88% amino acid sequence identity to rabbit UT2 and 64% identity to the recently cloned human erythrocyte urea transporter, HUT1l (Olives, B., P. Neav, P. Bailly, M. A. Hediger, G. Rousselet, J. P. Cartron, and P. Ripoch J. Biol. Chem. 1994. 269:31649-31652). Analysis of rat kidney mRNA revealed two transcripts of size 2.9 and 4.0 kb which had spatially distinct distributions. Northern analysis and in situ hybridization showed that the 4.0-kb transcript was primarily responsive to changes in the protein content of the diet whereas the 2.9-kb transcript was responsive to changes in the hydration state of the animal. These studies reveal that the expression levels of the two rUT2 transcripts are modulated by different pathways to allow fluid and nitrogen balance to be regulated independently. Our data provide important insights into the regulation of the renal urea transporter UT2 and provide a basis on which to refine our understanding of the urinary concentrating mechanism and its regulation.

International audience; In this study, we devised a cysteine-focused point mutation analysis of the chloride channel function of trout anion exchanger 1 (tAE1) expressed in X. laevis oocytes. Seven cysteines, belonging to the transmembrane domain of tAE1, were mutated into serines (either individually or in groups) and the effects of these mutations on the chloride conductance of injected oocytes were measured. We showed that three cysteines were essential for the functional expression of tAE1. Namely, mutations C462S, C583S and C588S reduced Cl À conductance by 68%, 52% and 83%, respectively, when compared to wild type tAE1. These residual conductances were still inhibited by 0.5 mM niflumic acid. Western blot experiments demonstrated that C462 was involved in protein expression onto the plasma membrane. A mutant devoid of this residue was unable to express onto the plasma membrane, especially if several other cysteines were missing: consequently, the cysteine-less mutant of tAE1 was not functional. C583 and C588 were involved in the channel function of tAE1 as shown by anion substitution experiments proving that selectivity of the mutated pore differs from the wild type one. On the contrary, they were not involved in the Cl À /HCO À 3 exchange function of tAE1, as demonstrated by intracellular pH measurements. These and several complementary mutations allow us to conclude that a mutant of tAE1 containing the sole C462 can drive a marginal Cl À current; however, the minimal configuration necessary to get optimal functional expression of the tAE1 chloride channel is that of a mutant containing unaffected residues C462, C583 and C588.

Conclusions Les microglies provenant de la moelle osseuse offrent un potentiel formidable en vue du traitement de la maladie d’Alzheimer, puisqu’elles sont attirees vers les proteines toxiques et sont en mesure de les eliminer. Il est interessant de noter que des proteines toxiques sont presentes dans d’autres maladies neurodegeneratives, telle que l’alpha-synucleine dans la maladie de Parkinson et la superoxyde dismutase dans la sclerose laterale amyotrophique. Les cellules souches de la moelle osseuse pourront ainsi se diriger vers les differents foyers inflammatoires provoques par ces proteines toxiques. Elles ont aussi l’avantage, contrairement aux autres types de cellules souches, d’infiltrer de facon naturelle les regions endommagees et de s’adapter aux conditions inflammatoires. De plus, le prelevement des cellules souches hematopoietiques evitera le rejet des cellules genetiquement modifiees, puisque le patient servira a la fois de donneur et de receveur. Il nous reste maintenant a trouver la facon de rendre ces cellules plus resistantes, a selectionner de meilleurs phagocytes par exemple, plus specifiques a la βA (ou a d’autres proteines toxiques), tout en evitant l’emballement de la reponse inflammatoire. Un tres beau defi scientifique et medical a relever ! ‡ Bone marrow stem cells to the rescue of Alzheimer’s disease REFERENCES

Feeding rats a low-protein (8%) diet (LPD) for 2 wk induces a facilitated urea transporter in rat initial inner medullary collecting ducts (IMCDs). To determine whether this is preceded by an increase in mRNA abundance, we designed degenerate polymerase chain reaction primers to the rabbit facilitated urea transporter (UT2; G. You, C. P. Smith, Y. Kanai, W.-S. Lee, M. Stelzner, and M. A. Hediger. Nature Lond. 365: 844–847, 1993) and amplified a 716-bp cDNA fragment to perform Northern analysis of the base or tip of rat inner medulla. In the base, the predominant transcript was a 2.9-kb band, which increased 55% after 1 wk on an LPD; there was no change in a 4-kb band. In the tip, the 4-kb band predominated, but neither band varied with an LPD. Next, we functionally characterized the induced urea transporter using microperfused initial IMCDs from rats fed an LPD for 2 wk. First, 100 pM arginine vasopressin (AVP) stimulated urea permeability (Purea); 10 nM AVP increased Purea further. Second, raising perfusate and bath osmolality to 690 mosmol/kgH2O (NaCl added) stimulated Purea; adding AVP (10 nM) increased Purea further. Third, thiourea reversibly inhibited AVP-stimulated Purea.(ABSTRACT TRUNCATED AT 250 WORDS)

International audience; The regulation of mRNA for aldose reductase, sorbitol dehydrogenase, and the Na+/Cl-/taurine cotransporter was studied with three in vivo models in which urinary concentration is reduced: Sprague-Dawley rats undergoing a water diuresis or fed a low-protein diet or Brattleboro rats. In Sprague-Dawley rats, 3 days of water diuresis reduced inner medullary aldose reductase mRNA abundance 6.5-fold compared with untreated rats, whereas sorbitol dehydrogenase and taurine cotransporter mRNA were unchanged. When water diuretic rats were acutely deprived of water, urine osmolality increased significantly after 4 h but aldose reductase mRNA did not increase until 12 h. Heat shock protein-70 mRNA was not increased by water deprivation. Second, in rats fed a low-protein diet for 3 wk, aldose reductase mRNA increased two-fold, whereas sorbitol dehydrogenase and taurine cotransporter mRNA were unchanged. Finally, in Brattleboro rats, urine osmolality and levels of aldose reductase and taurine cotransporter mRNA increased in response to 1 day of water deprivation, whereas sorbitol dehydrogenase mRNA was unchanged. Administering vasopressin (1 U/day) to Brattleboro rats for 8 days also increased urine osmolality and aldose reductase mRNA but did not alter sorbitol dehydrogenase or taurine cotransporter mRNA. This result is consistent with the hypothesis that changes in urine osmolality induce changes in aldose reductase mRNA abundance that are independent of vasopressin. It was concluded that, in rat inner medulla: (1) aldose reductase mRNA abundance varies with changes in water balance or dietary protein, whereas sorbitol dehydrogenase and taurine cotransporter mRNA do not; and (2) heat shock protein-70 mRNA abundance is not increased during acute osmotic stress.

Experiments were performed to characterize the P2 purinoceptor subtype responsible for cytoplasmic calcium mobilization in cells from the initial part of rabbit distal convoluted tubule (DCT). Free calcium concentration was measured in a DCT cell line (DC1) with the probe fura 2. Both ATP and UTP increased cytosolic Ca2+concentration ([Ca2+]i; EC503 and 6 μM, respectively). The order of potency for nucleotide analogs was ATP = UTP > adenosine 5′- O-[thiotriphosphate] ≫ ADP > UDP, which is consistent with the pharmacology of the P2Y2 receptor subtype. The increased [Ca2+]iresponses to ATP and UTP were strongly inhibited by suramin. Pretreatment of cells with pertussis toxin (PTX) attenuated the action of both nucleotides. Inhibition of phospholipase C with U-73122 totally blocked the [Ca2+]iresponse to ATP. Thus ATP- and UTP-stimulated [Ca2+]imobilization in DC1 cells appears to be mediated via the activation of P2Y2 purinoceptors coupled to a G protein mechanism that is partially sensitive to PTX. Calcium flux measurements showed that lanthanum- and nifedipine-sensitive calcium channels are involved in the [Ca2+]iresponse to ATP.