Effect of the p38 MAPK Inhibitor on the Expression of Metalloproteinases and Their Inhibitors during the Formation of Abdominal Adhesions

Background: The aim of this study was to assess the effect of blockade of the p38 mitogen-activated protein kinase (MAPK) on the expression of genes encoding metalloproteinases (MMPs) during the formation of adhesions in the abdominal cavity. Methods and Results: The experiments were carried out on male Wistar rats (n=75). The studies were carried out in two groups: Group 1 (control, n=35) – modelling the adhesive process; Group 2 (experimental, n=35) – modelling the adhesive process with intraperitoneal administration of Seroguard®—a prolonged form of the p38 MAPK inhibitor. The expression of the MMP1a, MMP2, MMP7, MMP9, and TIMP genes was assessed using real-time PCR. In the control group, overexpression of the MMP1a and MMP7 genes began from 6 hours after modeling the adhesive process, MMP9 – from Day 1, MMP2 – from Day 7 and persisted until the end of observation. With local blockade of p38 MAPK, the level of overexpression of genes encoding MMPs in the early stages was higher than in the control group (MMP1a – by Day 1; MMP7 – by 6 hours and Day 1, MMP9 – by 12 hours). From Day 3 to Day 14, the MMP1a and MMP7 expression in the experimental group was significantly lower than in the control group. Conclusion: The performed study demonstrated the involvement of different types of MMPs—collagenases (MMP1a), gelatinases (MMP2 and 9), matrilysins (MMP7)—in the rearrangement of the extracellular matrix during the process of adhesion formation in the abdominal cavity.

Overexpression of long non coding RNA OR3A4 is associated with altered p38 signalling, morphological and phenotypic changes, and reduced immunogenicity in Barretts Oesophagus

Background: Barretts Oesophagus (BO) presents a particular pathological dilemma, in that patients who have no dysplasia within their BO experience a small but significant risk of malignant progression each year. Screening programmes have attempted to reduce the mortality from BO associated oesophageal adenocarcinoma but cannot predict which BO patients will progress to invasive malignancy. We have previously identified the long non coding RNA, OR3A4, is differentially hypomethylated in progressive BO. We aimed to understand its role in BO pathogenicity Methods: The stable BO cell line CP-A, as well as the oesophageal adenocarcinoma cells line OE-33 was transfected with a lentiviral OR3A4 over-expression vector, and underwent high resolution microscopy, immunofluorescence, RT-qPCR, RNA sequencing, and targeted drug screening with the p38-MAPK inhibitor domipramod to understand the effects of OR3A4 expression on progression. We then compared progressive vs. non-progressive BO samples using quantitative multi-fluorophore (Vectra) immunohistochemistry. Results: Over-expression of OR3A4 in CP-A lines resulted in a hyperproliferative, dysplastic cellular phenotype, with strong over-expression of MAPK and anti-apoptotic pathways at the RNA and protein level, which was sensitive to the p38-MAPK inhibitor domipramod. Vectra immunohistochemistry demonstrated that progressive BO had reduced visibility associated with a reduction in CD8+ T-cells and CD68+ macrophages and reduced CD4+ T-cells in the stomal compartment. Conclusion: The overexpression of OR3A4, which we have previously shown is associated with progressive BO leads to a proliferative dysplastic cellular phenotype associated with increased, reversible MAPK signalling and loss of immune visibility.

Investigation of the Molecular Mechanisms by Which Endothelin-3 Stimulates Preadipocyte Growth

Endothelins induce many biological responses, and they are composed of three peptides: ET-1, ET-2, and ET-3. Reports have indicated that ET-1 regulates cell proliferation, adipogenesis, and other cell responses and that ET-3 stimulates the growth of gastrointestinal epithelial cells and melanocytes. However, the signalling pathways of ET3 that mediate the growth of fat cells are still unclear. Using 3T3-L1 white preadipocytes, we found that ET-3 induced increases in both cell number and BrdU incorporation. Pretreatment with an ETAR antagonist (but not an ETBR antagonist) blocked the ET-3-induced increases in both cell number and BrdU incorporation. Additionally, BQ610 suppressed the ET-3-induced increases in phosphorylation of AMPK, c-JUN, and STAT3 proteins, and pretreatment with specific inhibitors of AMPK, JNK/c-JUN, or JAK/STAT3 prevented the ET-3-induced increases in phosphorylation of AMPK, c-JUN, and STAT3, respectively. Neither p38 MAPK inhibitor nor PKC inhibitor altered the effects of ET-3 on cell growth. These data suggest that ET-3 stimulates preadipocyte growth through the ETAR, AMPK, JNK/c-JUN, and STAT3 pathways. Moreover, ET-3 did not alter HIB1B brown preadipocyte and D12 beige preadipocyte growth, suggesting a preadipocyte type-dependent effect. The results of this study may help explain how endothelin mediates fat cell activity and fat cell-associated diseases.

Catecholestradiol Activation of Adrenergic Receptors Induces Endometrial Cell Survival via p38 MAPK Signaling

Context Enhanced levels of catecholestradiols, 2-hydroxyestradiol (2-OHE2) or 4-hydroxyestradiol (4-OHE2), are reported in endometriosis. During gestation, catecholestradiol activation of adrenergic receptors (AR) elevates estrogen receptor (ER)-independent proliferation of uterine arterial endothelial cells. Objective To investigate β-AR-mediated catecholestradiol effects on human endometrial stromal cell (HESC) and epithelial cell survival in endometriosis. Design β-AR immunostaining of eutopic and ectopic endometria (n = 9). Assays for cell viability, 5-bromo-2′-deoxyuridine proliferation, apoptosis, quantitative PCR, and estrogenicity (alkaline phosphatase activity), as well as siRNA β-AR silencing and immunoblot analyses of cultured HESCs or Ishikawa cells treated with control or 2-OHE2 or 4-OHE2 ±β-AR antagonist or ±p38 MAPK inhibitor. Setting University research institution. Patients Women with or without endometriosis. Interventions None. Main Outcome Measures β-AR expression in eutopic vs ectopic endometria and regulation of HESC survival by 2-OHE2 and 4-OHE2. Results Eutopic and ectopic endometrial stromal and epithelial cells displayed β2-AR immunoreactivity with increased staining in the functionalis vs basalis layer (P < 0.05). Both 2-OHE2 and 4-OHE2 enhanced HESC and Ishikawa cell survival (P < 0.05), an effect abrogated by β-AR antagonist propranolol, but not ER antagonist ICI182,780. 2-OHE2 or 4-OHE2 failed to induce cell survival and estrogenic activity in ADRB2-silenced HESCs and in Ishikawa cells, respectively. Although 2-OHE2 inhibited apoptosis and BAX mRNA expression, 4-OHE2 induced proliferation and decreased apoptosis (P < 0.05). Both catecholestradiols elevated phospho-p38 MAPK levels (P < 0.05), which was blocked by propranolol, and p38 MAPK inhibitor reversed catecholestradiol-enhanced HESC survival. Conclusions Catecholestradiols increase endometrial cell survival by an ER-independent β-AR-mediated p38 MAPK activation, suggesting that agents blocking β-AR (e.g., propranolol) or inhibiting 2-OHE2- or 4-OHE2-generating enzymes (i.e., CYP1A1/B1) could treat endometriosis.

P0143OPPOSING EFFECTS OF P38 MAPK IN PODOCYTES ON ALDOSTERONE-INDUCED GLOMERULAR INJURY IN POD-GC-A CKO MICE

Background and Aims Previously, we demonstrated that uninephrectomized aldosterone-infused, high salt-fed podocyte-specific guanylyl cyclase-A (natriuretic peptide receptor 1) conditional KO (pod-GC-A cKO) mice exhibited glomerular injury and that pharmacological inhibition of p38 MAPK ameliorates podocyte damage. However, the effects of genetic deletion of p38 MAPK in podocytes of pod-GC-A cKO mice have been unclarified. Method We generated p38 MAPK(fl/fl);Nephrin-Cre (pod-p38 MAPK cKO) mice and p38 MAPK(fl/fl);GC-A(fl/fl);Nephrin-Cre (pod-p38MAPK/GC-A DKO) mice. For induction of glomerular injury, we treated them with aldosterone and high salt at 2 months of age for 3 weeks without nephrectomy (B-ALDO). In vitro, we examined the effect of p38 MAPK inhibitor in cultured human podocytes transfected with GC-A siRNA. Results B-ALDO-treated pod-p38 MAPK/GC-A DKO mice resulted in significant elevation of serum Cr (0.29 ± 0.04 mg/dl), massive albuminuria (42,660 ± 20,200 μg/mgCr) and severe foot process effacement in addition to intracapillary fibrin thrombi which indicated endothelial damage. Vehicle-treated DKO mice, B-ALDO-treated pod-GC-A cKO mice, and B-ALDO-treated pod-p38 MAPK cKO showed normal serum Cr levels (0.14 ± 0.01, 0.18 ± 0.02, 0.20 ± 0.01 mg/dl, respectively), mild increase of albuminuria (223 ± 6.5, 1,496 ± 592, 649 ± 303 μg/mgCr, respectively) and only segmental foot process effacement. Blood pressure was not elevated in either mutant mice compared with that of B-ALDO control mice. Furthermore, glomerular mRNA expressions of MCP-1, PAI-1, and FN were upregulated and that of VEGF-A was downregulated in DKO mice. In vitro, suppression of GC-A mRNA by siRNA in combination with p38 MAPK inhibitor downregulated VEGF mRNA in human cultured podocytes. Our previous works showed that pharmacological inhibition of p38 MAPK in the whole body ameliorated podocyte damage, whereas our current result showed that genetic deletion of p38 MAPK in podocytes aggravated renal injury. In order to explain the discrepancy in these results, we added an analysis of podocyte specific GC-A fl/fl p38 fl/+ cKO mice. Pod GC-A fl/fl p38 fl/+ cKO mice exhibited considerably milder renal damage than pod GC-A fl/fl p38 fl/fl double cKO mice. Conclusion Genetic complete p38 MAPK deletion in GC-A-nul podocytes exacerbated aldosterone-induced glomerular endothelial cell injury as well as podocytes, and resulted in renal dysfunction, probably through VEGF downregulation, whereas partial p38 MAPK inhibition in podocytes ameliorated aldosterone-induced glomerular injury in pod-GC-A cKO mice. These results suggest a certain level of p38 MAPK in podocytes is necessary to protect endothelial and epithelial cells from aldosterone-induced renal injury.

Heat Shock Protein 70 Protects the Heart from Ischemia/Reperfusion Injury through Inhibition of p38 MAPK Signaling

Background. Heat shock protein 70 (Hsp70) has been shown to exert cardioprotection. Intracellular calcium ([Ca2+]i) overload induced by p38 mitogen-activated protein kinase (p38 MAPK) activation contributes to cardiac ischemia/reperfusion (I/R) injury. However, whether Hsp70 interacts with p38 MAPK signaling is unclear. Therefore, this study investigated the regulation of p38 MAPK by Hsp70 in I/R-induced cardiac injury. Methods. Neonatal rat cardiomyocytes were subjected to oxygen-glucose deprivation for 6 h followed by 2 h reoxygenation (OGD/R), and rats underwent left anterior artery ligation for 30 min followed by 30 min of reperfusion. The p38 MAPK inhibitor (SB203580), Hsp70 inhibitor (Quercetin), and Hsp70 short hairpin RNA (shRNA) were used prior to OGD/R or I/R. Cell viability, lactate dehydrogenase (LDH) release, serum cardiac troponin I (cTnI), [Ca2+]i levels, cell apoptosis, myocardial infarct size, mRNA level of IL-1β and IL-6, and protein expression of Hsp70, phosphorylated p38 MAPK (p-p38 MAPK), sarcoplasmic/endoplasmic reticulum Ca2+-ATPase2 (SERCA2), phosphorylated signal transducer and activator of transcription3 (p-STAT3), and cleaved caspase3 were assessed. Results. Pretreatment with a p38 MAPK inhibitor, SB203580, significantly attenuated OGD/R-induced cell injury or I/R-induced myocardial injury, as evidenced by improved cell viability and lower LDH release, resulted in lower serum cTnI and myocardial infarct size, alleviation of [Ca2+]i overload and cell apoptosis, inhibition of IL-1β and IL-6, and modulation of protein expressions of p-p38 MAPK, SERCA2, p-STAT3, and cleaved-caspase3. Knockdown of Hsp70 by shRNA exacerbated OGD/R-induced cell injury, which was effectively abolished by SB203580. Moreover, inhibition of Hsp70 by quercetin enhanced I/R-induced myocardial injury, while SB203580 pretreatment reversed the harmful effects caused by quercetin. Conclusions. Inhibition of Hsp70 aggravates [Ca2+]i overload, inflammation, and apoptosis through regulating p38 MAPK signaling during cardiac I/R injury, which may help provide novel insight into cardioprotective strategies.