Molecular Mechanism of Stimulation of Na-K-ATPase by Leukotriene D4 in Intestinal Epithelial Cells

Na-K-ATPase provides a favorable transcellular Na gradient required for the functioning of Na-dependent nutrient transporters in intestinal epithelial cells. The primary metabolite for enterocytes is glutamine, which is absorbed via Na-glutamine co-transporter (SN2; SLC38A5) in intestinal crypt cells. SN2 activity is stimulated during chronic intestinal inflammation, at least in part, secondarily to the stimulation of Na-K-ATPase activity. Leukotriene D4 (LTD4) is known to be elevated in the mucosa during chronic enteritis, but the way in which it may regulate Na-K-ATPase is not known. In an in vitro model of rat intestinal epithelial cells (IEC-18), Na-K-ATPase activity was significantly stimulated by LTD4. As LTD4 mediates its action via Ca-dependent protein kinase C (PKC), Ca levels were measured and were found to be increased. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, also mediated stimulation of Na-K-ATPase like LTD4, while BAPTA-AM (Ca chelator) and calphostin-C (Cal-C; PKC inhibitor) prevented the stimulation of Na-K-ATPase activity. LTD4 caused a significant increase in mRNA and plasma membrane protein expression of Na-K-ATPase α1 and β1 subunits, which was prevented by calphostin-C. These data demonstrate that LTD4 stimulates Na-K-ATPase in intestinal crypt cells secondarily to the transcriptional increase of Na-K-ATPase α1 and β1 subunits, mediated via the Ca-activated PKC pathway.

Yoda1 and phosphatidylserine exposure in red cells from patients with sickle cell anaemia

Phosphatidylserine (PS) exposure is increased in red cells from sickle cell anaemia (SCA) patients. Externalised PS is prothrombotic and attractive to phagocytes and activated endothelial cells and thus contributes to the anaemic and ischaemic complications of SCA. The mechanism of PS exposure remains uncertain but it can follow increased intracellular Ca2+ concentration ([Ca2+]i). Normally, [Ca2+]i is maintained at very low levels but in sickle cells, Ca2+ permeability is increased, especially following deoxygenation and sickling, mediated by a pathway sometimes called Psickle. The molecular identity of Psickle is also unclear but recent work has implicated the mechanosensitive channel, PIEZO1. We used Yoda1, an PIEZO1 agonist, to investigate its role in sickle cells. Yoda1 caused an increase in [Ca2+]i and PS exposure, which was inhibited by its antagonist Dooku1 and the PIEZO1 inhibitor GsMTx4, consistent with functional PIEZO1. However, PS exposure did not necessitate an increase in [Ca2+]i. Two PKC inhibitors were also tested, chelerytherine chloride and calphostin C. Both reduced PS exposure whilst chelerytherine chloride also reduced Yoda1-induced increases in [Ca2+]i. Findings are therefore consistent with the presence of PIEZO1 in sickle cells, able to mediate Ca2+ entry but that PKC was also involved in both Ca2+ entry and PS exposure.

WNT11-Conditioned Medium Promotes Angiogenesis through the Activation of Non-Canonical WNT-PKC-JNK Signaling Pathway

Background: We demonstrated that the transduction of Wnt11 into mesenchymal stem cells (MSCs) (MSCWnt11) promotes these cells differentiation into cardiac phenotypes. In the present study, we investigated the paracrine effects of MSCWnt11 on cardiac function and angiogenesis. Methods and Results: Conditioned medium was collected from MSCWnt11 (CdMWnt11) and their control cells (CdMGFP). CdMWnt11, especially obtained from MSCWnt11 exposed to hypoxia, significantly promoted human umbilical vein endothelial cells (HUVECs) migration and increased capillary-like tube (CLT) formation, which was blocked by Wnt11 neutralizing antibody. Wnt11 protein was significantly higher in CdMWnt11 compared to that in CdMGFP. Directly treating HUVECs with recombinant Wnt11 protein significantly increased CLT formation, which was abrogated by treating cells with the JNK inhibitor SP600125, as well as the PKC inhibitor Calphostin-C. Moreover, the transfection of Wnt11 to HUVECs (HWnt11) significantly increased CLT formation and HUVEC migration, as well as upregulated p-pan-PKC and p-JNK expression. Injection of CdMWnt11 into the peri-infarct region in a rat acute myocardial infarction (AMI) model significantly improved cardiac function, reduced infarct size, and increased myocardial blood flow and blood vessel density in the ischemic area. Conclusion: Wnt11 released from MSCWnt11 increased angiogenesis and improved cardiac function via non-canonical Wnt-PKC-JNK dependent pathways.

Delta subfamily (version 2020.4) in the IUPHAR/BPS Guide to Pharmacology Database

PKCδ and PKCθ are PKC isoforms that are activated by diacylglycerol and may be inhibited by calphostin C, Gö 6983 and chelerythrine.

Further characterization of the synergistic activation mechanism of cationic channels by M2 and M3 muscarinic receptors in mouse intestinal smooth muscle cells

In mouse ileal myocytes, muscarinic receptor-mediated cationic current ( mIcat) occurs mainly through synergism of M2 and M3 subtypes involving Gi/o-type GTP-binding proteins and phospholipase C (PLC). We have further studied the M2/M3 synergistic pathway. Carbachol-induced mIcat was markedly depressed by YM-254890, a Gq/11 protein inhibitor. However, the mIcat was unaffected by heparin, calphostin C, or chelerythrine, suggesting that mIcat activation does not involve signaling molecules downstream of phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown. M2-knockout (KO) mice displayed a reduced mIcat (~10% of wild-type mIcat) because of the lack of M2-Gi/o signaling. The impaired mIcat was insensitive to neuropeptide Y possessing a Gi/o-stimulating activity. M3-KO mice also displayed a reduced mIcat (~6% of wild-type mIcat) because of the lack of M3-Gq/11 signaling, and the mIcat was insensitive to prostaglandin F2α possessing a Gq/11-stimulating activity. These results suggest the importance of Gq/11/PLC-hydrolyzed PIP2 breakdown itself in mIcat activation and also support the idea that the M2/M3 synergistic pathway represents a signaling complex consisting of M2-Gi/o and M3-Gq/11-PLC systems in which both G proteins are special for this pathway but not general in receptor coupling.

AML Cells Express Exclusively the Long Isoform of LEF1 and Are Highly Vulnerable to Blockage of LEF1-β-Catenin Binding

Despite progress in the understanding of the biology of acute myeloid leukemia (AML) in recent years, mortality of the disease is still high. One reason for this is drug resistance of leukemic stem cells (LSCs), which are responsible for leukemic growth and relapse. Thus, there is great interest in understanding factors driving LSCs and in key differences between normal hematopoietic stem cells (HSC) and their leukemic counterparts. LEF1 acts via the canonical Wnt pathway by interacting with β-catenin (long isoform). Of note, a natural isoform of Lef1 is expressed in hematopoiesis, which lacks the β-catenin binding domain at the N-terminus and whose function is not well defined in normal and leukemic hematopoiesis (short isoform). Here we demonstrate in a cohort of 111 AML primary patient samples with normal karyotype, that exclusively the long isoform of LEF1 is expressed. These analyses were extended to functionally validated LSC populations, using the NSG xenograft model, and confirmed the highly predominant expression of the long isoform of LEF1 and the virtual lack of expression of the short isoform. This stood in clear contrast to normal HSCs: in highly purified CD34-/CD38-/CD93high HSCs, categorized as most immature human quiescent HSC population, as well as CD34+/CD49f+/CD90+ and CD34+/CD49f+/CD90- human HSCs, no expression of the long isoform could be detected with an exclusive expression of the short isoform of LEF1. In multipotent progenitors MPPs (CD34+/CD49f-/CD90- ) there was a shift towards expression of both isoforms. Thus, LSCs show an inverse expression pattern of LEF1 isoforms compared to their normal counterparts. To understand the function of the long versus short isoform, we cloned the murine long isoform and the Lef1 isoform lacking the ß-catenin binding domain at the N-terminus into retroviral expression vectors and transduced primary murine bone marrow cells. In line with our observation of the exclusive expression of the long isoform of LEF1 in human AML stem cells, we previously showed that expression of the long isoform induces AML in transplanted mice (Petropoulos et al., JEM 2008). In contrast, expression of the short isoform did not have any major effect at the level of repopulating stem cells as determined by quantifying CRU frequency in limit dilution transplantation assays. Furthermore, ChiP-Seq analyses showed predominant binding of the short isoform on genes associate re-entry of HSCs into quiescence such as like CD81 and Wnt5a, in line with our observation of exclusive expression of the short isoform in the human CD34-/CD38-/CD93high HSC population. Based on the observation that AML cells exclusively express the long isoform, which acts via β-catenin binding, we hypothesized that AML cells would be particularly vulnerable to drugs blocking the binding of LEF1-β-catenin binding. The small molecule inhibitors Cercosporin and Calphostin C (100nM), previously shown to block LEF1-β-catenin binding in CLL cells, reduced colony growth of the AML cell line THP1 by 100 % (p<0,0001) and impaired engraftment in NSG mice (26d control vs 32d Cercosporin 100nM, p=0,0013, and 53d Calphostin C, p= 0,0022). In contrast, normal CD34+ cord blood cells were much less vulnerable to these compounds (0,2% reduction of CFC growth with Cercosporin, 8% reduction in Calphostin C, not significant). Taken together, these data point to distinct LEF1 isoform expression in AML, thereby creating high vulnerability towards blockage of LEF1-β-catenin binding and forming a potentially targetable Achilles' heel at the level of leukemic stem cells. Disclosures Metzeler: Novartis: Consultancy; Celgene: Consultancy, Research Funding. Buske:Roche: Honoraria, Research Funding; Bayer: Research Funding; Janssen: Honoraria, Research Funding.

168 THE DEVELOPMENTAL POTENTIAL OF PARTHENOGENETIC EMBRYOS IS AFFECTED BY PROLACTIN DURING THE PROLONGED CULTURE OF BOVINE CUMULUS-ENCLOSED OOCYTES

The technique of somatic cell nuclear transfer includes a delayed activation of reconstituted oocytes (4–6 h after IVM). The prolonged culture of mammalian oocytes is known to be associated with aging of the ova leading to a decline in their quality and developmental capacity. The aim of the present research was to study effects of 2 closely related hormones, prolactin (PRL) and growth hormone (GH), during the prolonged culture of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-enclosed oocytes (CEO) were cultured for 16 h in TCM 199 containing 10% FCS, 10 μg mL−1 of porcine FSH, and 10 μg mL−1 of ovine LH. After 16 h of maturation, CEO or denuded oocytes were transferred to the fresh medium consisting of TCM 199 supplemented with 10% FCS and cultured for 12 h in the absence (Control) or in presence of 50 ng mL−1 of bovine PRL or 10 ng mL−1 of recombinant bovine GH or protein kinase inhibitors. The following inhibitors were used: (1) PP2 (an inhibitor of Src-family tyrosine kinases, 10 mM), (2) triciribine (an inhibitor of Akt kinase, 25 μM), and (3) calphostin C (a protein kinase C inhibitor, 0.5 μM). After the prolonged culture for 12 h, oocytes were activated by sequential treatment with ionomycin (5 μM for 5 min) immediately followed by 6-dimethylaminopurine (2 mM for 4 h). Activated oocytes were cultured in CR1aa medium until Day 5 postactivation and then transferred to the same medium supplemented with 5% FCS and cultured up to Day 7. The cleavage and blastocyst rates were assessed at Day 2 and 7, respectively. The data from 4 to 6 replicates (106–169 oocytes per treatment) were analysed by ANOVA. After the prolonged culture of CEO, the cleavage rate did not differ between the control and the hormone-treated groups, varying from 64.3 to 70.1%. Meanwhile, the blastocyst yield in the control group (12.5 ± 1.6%) was lower than in the PRL group (21.5 ± 2.4%, P < 0.05), but was similar to that in the GH group (13.9 ± 1.8%). Furthermore, the developmental capacity of cultured denuded oocytes was unaffected by both GH and PRL. The enhancing effect of PRL on the yield of parthenogenetic blastocysts derived from CEO was also observed in the presence of PP2 (26.8 ± 2.4 v. 15.7 ± 2.0%; P < 0.01). By contrast, calphostin C abolished this effect of PRL, although it did not affect the developmental potential of CEO in the control medium. At the same time triciribine reduced the blastocyst rate both in the control and PRL-treated groups (from 16.5 ± 1.8 to 8.1 ± 0.8% and from 24.3 ± 1.6 to 16.3 ± 2.4%, respectively; P < 0.05). Our data indicate that PRL can support the developmental potential of parthenogenetic embryos by affecting bovine oocytes during the prolonged culture, with the effect being mediated by cumulus cells and achieved via activation of protein kinase C. This research was supported by the Federal Agency for Scientific Organizations and RFBR (project No. 15-08-99473).

PKC-α-dependent augmentation of cAMP and CREB phosphorylation mediates the angiotensin II stimulation of renin in the collecting duct

In contrast to the negative feedback of angiotensin II (ANG II) on juxtaglomerular renin, ANG II stimulates renin in the principal cells of the collecting duct (CD) in rats and mice via ANG II type 1 (AT1R) receptor, independently of blood pressure. In vitro data indicate that CD renin is augmented by AT1R activation through protein kinase C (PKC), but the exact mechanisms are unknown. We hypothesize that ANG II stimulates CD renin synthesis through AT1R via PKC and the subsequent activation of cAMP/PKA/CREB pathway. In M-1 cells, ANG II increased cAMP, renin mRNA (3.5-fold), prorenin, and renin proteins, as well as renin activity in culture media (2-fold). These effects were prevented by PKC inhibition with calphostin C, PKC-α dominant negative, and by PKA inhibition. Forskolin-induced increases in cAMP and renin expression were prevented by calphostin C. PKC inhibition and Ca2+ depletion impaired ANG II-mediated CREB phosphorylation and upregulation of renin. Adenylate cyclase 6 (AC) siRNA remarkably attenuated the ANG II-dependent upregulation of renin mRNA. Physiological activation of AC with vasopressin increased renin expression in M-1 cells. The results suggest that the ANG II-dependent upregulation of renin in the CD depends on PKC-α, which allows the augmentation of cAMP production and activation of PKA/CREB pathway via AC6. This study defines the intracellular signaling pathway involved in the ANG II-mediated stimulation of renin in the CD. This is a novel mechanism responsible for the regulation of local renin-angiotensin system in the distal nephron.

PKC-Dependent Signaling Pathways within PAG and Thalamus Contribute to the Nitric Oxide-Induced Nociceptive Behavior

Nitric oxide (NO) is an important molecule involved in nociceptive processing in the central nervous system. The release of NO within the spinal cord has long been implicated in the mechanisms underlying exaggerated pain sensitivity, and administration of NO donors can induce hyperalgesia. To elucidate the supraspinal mechanism responsible for NO-induced nociceptive hypersensitivity, we investigated the modulation of protein kinase C (PKC) and downstream effectors following treatment with the NO donors nitroglycerin and sodium nitroprusside. Both compounds induced a prolonged cold allodynia and heat hyperalgesia, increased levels of c-Fos and IL-1β, and activated NF-κB within periaqueductal grey matter and thalamus. Simultaneously, an increased expression and phosphorylation of PKC γ and ε were detected. To clarify the cellular mechanism involved in the NO-induced hypernociception, we examined the expression of transcription factors that act as PKC downstream effectors. A dramatic hyperphosphorylation of CREB and STAT1 was observed. The i.c.v. administration of the PKC blocker calphostin C prevented the NO-induced hypernociception, the hyperphosphorylation of CREB and STAT1, and partially reduced NF-κB activation. Conversely, the increase of IL-1β was unmodified by calphostin C. These results suggest the relevance of cerebral PKC-mediated CREB and STAT1 activation in the NO donor-induced nociceptive behavior.