Conversion of SB 203580-insensitive MAP kinase family members to drug-sensitive forms by a single amino-acid substitution

Background: Specific inhibitors of protein kinases have great therapeutic potential, but the molecular basis underlying their specificity is only poorly understood. We have investigated the drug SB 203580 which belongs to a class of pyridinyl imidazoles that inhibits the stress-activated protein (SAP) kinases SAPK2a/p38 and SAPK2b/p38β2 but not other mitogen-activated protein kinase family members. Like inhibitors of other protein kinases, SB 203580 binds in the ATP-binding pocket of SAPK2a/p38. Results: The SAP kinases SAPK1γ/JNK1, SAPK3 and SAPK4 are not inhibited by SB 203580, because they have methionine in the position equivalent to Thr106 in the ATP-binding region of SAPK2a/p38 and SAPK2b/p38β2. Using site-directed mutagenesis of five SAP kinases and the type I and type II TGFβ receptors, we have established that for a protein kinase to be inhibited by SB 203580, the sidechain of this residue must be no larger than that of threonine. Sensitivity to inhibition by SB 203580 is greatly enhanced when the sidechain is even smaller, as in serine, alanine or glycine. Thus, the type I TGFβ receptor, which has serine at the position equivalent to Thr106 of SAPK2a/p38 and SAPK2b/p38β2, is inhibited by SIB 203580. Conclusions: These findings explain how drugs that target the ATP-binding site can inhibit protein kinases specifically, and show that the presence of threonine or a smaller amino acid at the position equivalent to Thr106 of SAPK2a/p38 and SAPK2b/p38β2 is diagnostic of whether a protein kinase is sensitive to the pyridinyl imidazole class of inhibitor.     

The p38 mitogen-activated protein kinase is involved in stress-induced phospholipase D activation in vascular smooth muscle cells

Oxidative stress has been implicated in mediation of vascular disorders. Earlier study showed that the exposure of vascular smooth muscle cells (VSMC) to pervanadate (hydrogen peroxide plus orthovanadate) resulted in the accumulation of [3H]phosphatidylbutanol. In this study, the effect of pervanadate on the activation of p38 mitogen-activated protein kinase (p38 MAPK) was studied in the VSMC. Pervanadate treatment activated p38 MAPK in a dose-and time-dependent manner. Interestingly, specific inhibition of p38 MAPK with SB203580 attenuated pervanadate-induced PLD activation. This correlates with the finding that expression of dominant negative mutants of MKK3/6 inhibited the PLD activation. SB203580 pretreatment also inhibited other cellular stressors (i.e. high osmolarity and UV light)-induced PLD activation. The possible correlationship of p38 MAPK activation with PKC was examined since PKC is reported to be involved in the pervanadate-induced PLD activation. Calphostin C, a PKC inhibitor, suppressed pervanadate-induced p38 MAPK and PLD activation in a dose-dependent manner. These results suggest that PKC-p38 MAPK may represent an upstream pathway of PLD in the signal transduction of cellular stress.     

Activation of epidermal growth factor receptor is responsible for pervanadate-induced phospholipase D activation

Pervanadate, a complex of vanadate and H(2)O(2), has an insulin mimetic effect, and acts as an inhibitor of protein tyrosine phosphatase. Pervanadate-induced phospholipase D (PLD) activation is known to be dependent on the tyrosine phosphorylation of cellular proteins and protein kinase C (PKC) activation, and yet underlying molecular mechanisms are not clearly understood. Here, we investigated the signaling pathway of pervanadate-induced PLD activation in Rat2 fibroblasts. Pervanadate increased PLD activity in dose- and time- dependent manner. Protein tyrosine kinase inhibitor, genistein, blocked PLD activation. Interestingly, AG-1478, a specific inhibitor of the tyrosine kinase activity of epidermal growth factor receptor (EGFR) blocked not only the PLD activation completely but also phosphorylation of p38 mitogen-activated protein kinase (MAPK). However, AG-1295, an inhibitor specific for the tyrosine kinase activity of pletlet drived growth factor receptor (PDGFR) did not show any effect on the PLD activation by pervanadate. We further found that pervanadate increased phosphorylation levels of p38, extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK). SB203580, a p38 MAPK inhibitor, blocked the PLD activation completely. However, the inhibitions of ERK by the treatment of PD98059 or of JNK by the overexpression of JNK interacting peptide JBD did not show any effect on pervanadate-induced PLD activation. Inhibition or down-regulation of PKC did not alter the pervanadate-induced PLD activation in Rat2 cells. Thus, these results suggest that pervanadate-induced PLD activation is coupled to the transactivation of EGFR by pervanadate resulting in the activation of p38 MAP kinase.     

Downstream components of RhoA required for signal pathway of superoxide formation during phagocytosis of serum opsonized zymosans in macrophages

Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum- (SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat-C3 blocked superoxide production, suggesting that RhoA is essentially involved in superoxide formation during phagocytosis of SOZ. Conversely SOZ activated both RhoA and Rac1/2. Inhibition of RhoA-activated kinase (ROCK), an important downstream effector of RhoA, by Y27632 and myosin light chain kinase (MLCK) by ML-7 abrogated superoxide production by SOZ. Extracellular signaling-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were activated during phagocytosis of SOZ, and Tat-C3 and SB203580 reduced ERK1/2 and p38 MAPK activation, suggesting that RhoA and p38 MAPK may be upstream regulators of ERK1/2. Inhibition of ERK1/2, p38 MAPK, phosphatidyl inositol 3-kinase did not block translocation of RhoA to membranes, suggesting that RhoA is upstream to these kinases. Inhibition of RhoA by Tat-C3 blocked phosphorylation of p47(PHOX). Taken together, RhoA, ROCK, p38MAPK, ERK1/2, and p47(PHOX) may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.     

Involvement of MAPK pathway in hypoxia-induced up-regulation of urokinase plasminogen activator receptor in a human prostatic cancer cell line, PC3MLN4

Clinical studies have shown that tumor hypoxia is associated with invasive growth and metastasis and may be an important prognostic factor adversely influencing survival in patients with tumors. To investigate the mechanisms involved in hypoxia-induced invasive growth and metastasis, hypoxia-mediated urokinase plasmalogen activator receptor (uPAR) expression, cellular invasiveness, and mitogen activated protein kinase (MAPK) activation were measured in a prostate cancer cell line, PC3MLN4. The levels of uPAR expression and cellular invasiveness were increased in hypoxic cells. Hypoxia-induced cellular invasiveness was blocked by an anti-uPAR monoclonal antibody. Phosphorylations of ERK and p38 kinases were also more extensive in hypoxic cells than in normoxic cells. Hypoxia-induced uPAR up-regulation was inhibited by pre-treatments with a specific inhibitor of MEK, PD98059 and a specific inhibitor of p38 MAP kinase, SB203580. Cell growth also increased in hypoxic cells. From these results, hypoxia increased tumor cell invasion by up-regulating uPAR expression, which might be mediated through ERK and p38 kinase signaling pathways in PC3MLN4 prostate cancer cell line.     

Interleukin-1beta stimulates matrix metalloproteinase-2 expression via a prostaglandin E2-dependent mechanism in human chondrocytes

IL-1beta is known promote cyclooxygenase-2 (COX- 2) and matrix metalloproteinase-2 (MMP-2) expression. This study focuses on the characterization of the signaling cascade associated with IL-1beta-induced matrix metalloproteinase-2 (MMP-2) regulation in human chondrocytes. The decrease in collagen levels in the conditioned media was prevented by a broad spectrum MMP inhibitor, suggesting that IL-1beta promotes the proteolytic process leading to MMP-2 activation. IL-1beta-related MMP-2 expression was found to be dependent on prostaglandin E2 (PGE2) production. In addition, the induction of COX-2 and MMP-2 was inhibited by the pretreatment of chondrocytes with a SB203580 or Ro 31-8220, indicating the involvement of protein kinase C (PKC) or p38 mitogen-activated protein kinase (MAPK). However, there is no cross-talk between PKC and p38 MAPK in the IL-1beta-induced MMP-2 activation. Taken together, these results demonstrated that IL-1beta induces MMP-2 expression through the PGE2-dependent mechanism in human chondrocytes.     

Rottlerin enhances IL-1β-induced COX-2 expression through sustained p38 MAPK activation in MDA-MB-231 human breast cancer cells

Cyclooxygenase-2 (COX-2) is an important enzyme in inflammation. In this study, we investigated the underlying molecular mechanism of the synergistic effect of rottlerin on interleukin1β (IL-1β)-induced COX-2 expression in MDA-MB-231 human breast cancer cell line. Treatment with rottlerin enhanced IL-1β-induced COX-2 expression at both the protein and mRNA levels. Combined treatment with rottlerin and IL-1β significantly induced COX-2 expression, at least in part, through the enhancement of COX-2 mRNA stability. In addition, rottlerin and IL-1β treatment drove sustained activation of p38 Mitogen-activated protein kinase (MAPK), which is involved in induced COX-2 expression. Also, a pharmacological inhibitor of p38 MAPK (SB 203580) and transient transfection with inactive p38 MAPK inhibited rottlerin and IL-1β-induced COX-2 upregulation. However, suppression of protein kinase C δ (PKC δ) expression by siRNA or overexpression of dominant-negative PKC δ (DN-PKC-δ) did not abrogate the rottlerin plus IL-1β-induced COX-2 expression. Furthermore, rottlerin also enhanced tumor necrosis factor-α (TNF-α), phorbol myristate acetate (PMA), and lipopolysaccharide (LPS)-induced COX-2 expression. Taken together, our results suggest that rottlerin causes IL-1β-induced COX-2 upregulation through sustained p38 MAPK activation in MDA-MB-231 human breast cancer cells.     

Catechins inhibit angiotensin II-induced vascular smooth muscle cell proliferation via mitogen-activated protein kinase pathway

Catechins, components of green tea, reduce the incidence of cardiovascular diseases such as atherosclerosis. Angiotensin II (Ang II) is highly implicated in the proliferation of vascular smooth muscle cells (VSMC), resulting in atherosclerosis. The acting mechanisms of the catechins remain to be defined in the proliferation of VSMC induced by Ang II. Here we report that catechin, epicatechin (EC), epicatechingallate (ECG) or epigallocatechingallate (EGCG) significantly inhibits the Ang II-induced [3H]thymidine incorporation into the primary cultured rat aortic VSMC. Ang II increases the phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK 1/2), c-jun-N-terminal kinase 1/2 (JNK 1/2), or p38 mitogen-activated protein kinases (MAPKs) and mRNA expression of c-jun and c-fos. The EGCG pretreatment inhibits the Ang II-induced phosphorylation of ERK 1/2, JNK 1/2, or p38 MAPK, and the expression of c-jun or c-fos mRNA. U0126, a MEK inhibitor, SP600125, a JNK inhibitor, or SB203580, a p38 inhibitor, attenuates the Ang II-induced [3H]thymidine incorporation into the VSMC. In conclusion, catechins inhibit the Ang II-stimulated VSMC proliferation via the inhibition of the Ang II-stimulated activation of MAPK and activator protein-1 signaling pathways. The antiproliferative effect of catechins may be associated with the reduced risk of cardiovascular diseases by the intake of green tea. Catechins may be useful in the development of prevention and therapeutics of vascular diseases.     

Activation of JNK and p38 in rat hippocampus after kainic acid induced seizure

Kainic acid, an analogue of glutamate, causes limbic seizures and induces cell death in the rat brain. We examined the activation of MAPK family kinases; ERKs, JNKs and p38 kinase in rat hippocampus after KA treatment. Activation of all three kinases were observed at 30 min after the treatment, but, in contrary to ERK phosphorylation, which lasted up to 3 h, the phosphorylation of JNK and p38 returned to the basal level by 2 h. The phosphorylation of' upstream kinases for the MAPK family was distinct. The phosphorylation of MEK1 clearly increased at 30 min but diminished rapidly thereafter. The phosphorylation of MKK6 was also increased but reached peak at 2 h after KA treatment. However, the phosphorylation of other upstream kinases, SEK1 and MKK3, gradually decreased to 3 h after KA treatment. These results indicate that the KA activates all of the three MAPK family kinases with different time patterns and suggest the possibility that MKK3 and MKK6, and SEK1 may not be the upstream kinases for p38 and JNK in rat hippocampus.     

PKCalpha induces differentiation through ERK1/2 phosphorylation in mouse keratinocytes

Epidermal keratinocyte differentiation is a tightly regulated stepwise process that requires protein kinase C (PKC) activation. Studies on cultured mouse keraitnocytes induced to differentiate with Ca2+ have indirectly implicated the involvement of PKCa isoform. When PKCalpha was overexpressed in undifferentiated keratinocytes using adenoviral system, expressions of differentiation markers such as loricrin, filaggrin, keratin 1 (MK1) and keratin 10 (MK10) were increased, and ERK1/2 phosphorylation was concurrently induced without change of other MAPK such as p38 MAPK and JNK1/2. Similarly, transfection of PKCalpha kinase active mutant (PKCalpha- CAT) in the undifferentiated keratinocyte, but not PKCbeta-CAT, also increased differentiation marker expressions. On the other hand, PKCalpha dominant negative mutant (PKCbeta-KR) reduced Ca2+ -mediated differentiation marker expressions, while PKCbeta-KR did not, suggesting that PKCalpha is responsible for keratinocyte differentiation. When downstream pathway of PKCalpha in Ca2+ -mediated differentiation was examined, ERK1/2, p38 MAPK and JNK1/2 phosphorylations were increased by Ca2+ shift. Treatment of keratinocytes with PD98059, MEK inhibitor, and SB20358, p38 MAPK inhibitor, before Ca2+ shift induced morphological changes and reduced expressions of differentiation markers, but treatment with SP60012, JNK1/2 inhibitor, did not change at all. Dominant negative mutants of ERK1/2 and p38 MAPK also inhibited the expressions of differentiation marker expressions in Ca2+ shifted cells. The above results indicate that both ERK1/2 and p38 MAPK may be involved in Ca2+ -mediated differentiation, and that only ERK1/2 pathway is specific for PKCalpha-mediated differentiation in mouse keratinocytes.     

Regulation of hepatocyte growth factor-mediated urokinase plasminogen activator secretion by MEK/ERK activation in human stomach cancer cell lines

The regulatory mechanisms for the proliferation and the particular invasive phenotypes of stomach cancers are not still fully understood. Up-regulations of hepatocytes growth factor (HGF), its receptor (c-Met), and urokinase-type plasminogen activator (uPA) are correlated with the development and metastasis of cancers. In order to investigate roles of HGF/c-Met signaling in tumor progression and metastasis in stomach cancers, we determined effects of a specific MEK1 inhibitor (PD098059) and a p38 kinase inhibitor (SB203580) on HGF-mediated cell proliferation and uPA expression in stomach cancer cell lines (NUGC-3 and MKN-28). HGF treatment induced the phosphorylations of ERK and p38 kinase in time- and dose- dependent manners. Pre-treatment with PD098059 reduced HGF-mediated cell proliferation and uPA secretion. In contrast, SB203580 pre-treatment enhanced cell proliferation and uPA secretion due to induction of ERK phosphorylation. Stable expression of dominant negative-MEK1 in NUGC-3 cells showed a decrease in HGF-mediated uPA secretion. These results suggest that interaction of a MEK/ERK and a p38 kinase might play an important role in proliferation and invasiveness of stomach cancer cells.     

The mechanism of Raf activation through dimerization

Raf, a threonine/serine kinase in the Raf/MEK/ERK pathway, regulates cell proliferation. Raf''s full activation requires dimerization. Aberrant activation through dimerization is an important therapeutic target. Despite its clinical importance, fundamental questions, such as how the side-to-side dimerization promotes the OFF-to-ON transition of Raf''s kinase domain and how the fully activated ON-state kinase domain is stabilized in the dimer for Raf signaling, remain unanswered. Herein, we decipher an atomic-level mechanism of Raf activation through dimerization, clarifying this enigma. The mechanism reveals that the replacement of intramolecular π–π stacking by intermolecular π–π stacking at the dimer interface releases the structural constraint of the αC-helix, promoting the OFF-to-ON transition. During the transition, the inhibitory hydrophobic interactions were disrupted, making the phosphorylation sites in A-loop approach the HRD motif for cis-autophosphorylation. Once fully activated, the ON-state kinase domain can be stabilized by a newly identified functional N-terminal basic (NtB) motif in the dimer for Raf signaling. This work provides atomic level insight into critical steps in Raf activation and outlines a new venue for drug discovery against Raf dimerization.

We decipher an atomic-level mechanism of Raf activation through dimerization, revealing that the disruption of intramolecular π–π stacking at the dimer interface promotes the OFF-to-ON transition.     

p38 MAPK and ERK activation by 9-cis-retinoic acid induces chemokine receptors CCR1 and CCR2 expression in human monocytic THP-1 cells

9-cis-Retinoic acid (9CRA) plays an important role in the immune response; this includes cytokine production and cell migration. We have previously demonstrated that 9CRA increases expression of chemokine receptors CCR1 and CCR2 in human monocytes. To better understand how 9CRA induces CCR1 and CCR2 expression, we examined the contribution of signaling proteins in human monocytic THP-1 cells. The mRNA and surface protein up-regulation of CCR1 and CCR2 in 9CRA-stimulated cells were weakly blocked by the pretreatment of SB202190, a p38 MAPK inhibitor, and PD98059, an upstream ERK inhibitor. Activation of p38 MAPK and ERK1/2 was induced in both a time and dose-dependent manner after 9CRA stimulation. Both p38 MAPK and ERK1/2 phosphorylation peaked at 2 h after a 100 nM 9CRA treatment. 9CRA increased calcium influx and chemotactic activity in response to CCR1-dependent chemokines, Lkn-1/CCL15, MIP-1alpha/CCL3, and RANTES/CCL5, and the CCR2-specific chemokine, MCP-1/CCL2. Both SB202190 and PD98059 pretreatment diminished the increased calcium mobilization and chemotactic ability due to 9CRA. SB202190 inhibited the expression and functional activities of CCR1 and CCR2 more effectively than did PD98059. Therefore, our results demonstrate that 9CRA transduces the signal through p38 MAPK and ERK1/2 for CCR1 and CCR2 up-regulation, and may regulate the pro-inflammatory process through the p38 MAPK and ERK-dependent signaling pathways.     

Prunus yedoensis Matsum. stimulates glucose uptake in L6 rat skeletal muscle cells by activating AMP-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways

Prunus yedoensis Matsum. is used as a medicinal plant to alleviate symptoms of diabetes; however, the molecular mechanism underlying its antihyperglycaemic activity is unknown. In this study, we investigated the antihyperglycaemic effects of P. yedoensis and its molecular mechanism. Prunus yedoensis leaf extract (PLE) increased the glucose uptake of phosphorylatinginsulin receptor substrate (IRS)-1, 3'-phosphoinositide-dependent kinase (PDK)-1 and Akt PLE, and also increased the phosphorylation of AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (p38 MAPK). PLE-stimulated glucose uptake was blocked by an AMPK inhibitor (Compound C) and a p38 MAPK inhibitor (SB203580). Inhibition of AMPK activity reduced p38 MAPK phosphorylation, whereas the inhibition of p38 MAPK activity did not affect AMPK phosphorylation. Pretreatment with the phosphatidylinositol 3-kinase inhibitor LY294002 and Compound C reduced PLE-stimulated glucose uptake. Our results demonstrate that PLE stimulated glucose uptake by activating both insulin signalling and AMPK-p38 MAPK pathways. PLE shows potential as a natural antihyperglycaemic agent.     

p38 mitogen-activated protein kinase activation by ultraviolet A radiation in human dermal fibroblasts

UVA radiation penetrates deeply into the skin reaching both the epidermis and the dermis. We thus investigated the effects of naturally occurring doses of UVA radiation on mitogen-activated protein kinase (MAPK) activities in human dermal fibroblasts. We demonstrated that UVA selectively activates p38 MAPK with no effect on extracellular-regulated kinases (ERK1-ERK2) or JNK-SAPK (cJun NH2-terminal kinase-stress-activated protein kinase) activities. We then investigated the signaling pathway used by UVA to activate p38 MAPK. L-Histidine and sodium azide had an inhibitory effect on UVA activation of p38 MAPK, pointing to a role of singlet oxygen in transduction of the UVA effect. Afterward, using prolonged cell treatments with growth factors to desensitize their signaling pathways or suramin to block growth factor receptors, we demonstrated that UVA signaling pathways shared elements with growth factor signaling pathways. In addition, using emetine (a translation inhibitor altering ribosome functioning) we detected the involvement of ribotoxic stress in p38 MAPK activation by UVA. Our observations suggest that p38 activation by UVA in dermal fibroblasts involves singlet oxygen-dependent activation of ligand-receptor signaling pathways or ribotoxic stress mechanism (or both). Despite the activation of these two distinct signaling mechanisms, the selective activation of p38 MAPK suggests a critical role of this kinase in the effects of UVA radiation.     

Role of p38 mitogen-activated protein kinase and caspases in UV-B-induced apoptosis of murine peritoneal macrophages

The mechanisms of ultraviolet-B (UV-B)-induced apoptosis and the role of p38 mitogen-activated protein kinase (MAPK) were investigated in murine peritoneal macrophages. Exposure of murine peritoneal macrophages to UV-B irradiation induced rapid apoptosis concurrent with DNA fragmentation and activation of caspase-3 but did not activate caspase-1. UV-B irradiation (100 mJ/cm2) also induced expression of phospho-p38 and -c-Jun N-terminal kinase (JNK) MAPK; however, no significant expression of phospho-p42/44 was observed 120 min after exposure. Pretreatment of macrophages with a p38 MAPK inhibitor, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole (SB202190), and a caspase-3 inhibitor, N-acetyl-Asp-Glu-Val-Asp-CHO, suppressed UV-B irradiation-induced apoptosis as observed by DNA laddering and DNA fragmentation estimation quantitatively. Pretreatment with caspase-1 inhibitor, N-acetyl-Tyr-Val-Ala-Asp-CHO, had no effect. UV-B-induced caspase-3 activation resulted in the cleavage of poly-(ADP-ribose) polymerase (PARP), which was inhibited by the caspase-3 inhibitor. SB202190 pretreatment also prevented activation of caspase-3 and the cleavage of PARP. However, the caspase-3 and -1 inhibitors did not affect UV-B-induced expression of phospho-p38 and -JNK. These results suggest that activation of p38 MAPK upstream of caspases might play an important role in the apoptotic process of macrophages exposed to UV-B irradiation.     

Role of p38 MAPKs in Hypericin Photodynamic Therapy-induced Apoptosis of Nasopharyngeal Carcinoma Cells

The present study aims to determine the role of mitogen-activated protein kinases (MAPKs) in hypericin-mediated photodynamic therapy (HY-PDT)-induced apoptosis of the HK-1 nasopharyngeal carcinoma (NPC) cells. HY-PDT was found to induce proteolytic cleavage of procaspase-9 and -3 in HK-1 cells. Apoptotic nuclei were observed at 6 h after PDT whereas B-cell leukemia/lymphoma-2-associated-X-protein (Bax) translocation and formation of Bax channel is responsible for the cell death. Increase in phosphorylation of p38 MAPKs and c-Jun N-terminal kinase 1/2 (JNK1/2) was detected at 15–30 min after HY-PDT. The appearance of phosphorylated form of p38 MAPKs and JNK1/2 was inhibited by the singlet oxygen scavenger l -histidine. HY-PDT-induced cell death was enhanced by the chemical inhibitors for p38 MAPKs (SB202190 and SB203580), but not by the JNKs inhibitor SP600125. Knockdown of the p38α and p38β MAPK isoforms by small interfering RNA (siRNA) are more effective than the p38δ in enhancing PDT-induced cell death. Augmentation of apoptosis by p38α or p38β knockdown is also correlated with the increased proteolytic cleavage of procaspase-9 after HY-PDT treatment. Our results suggested that HY-PDT activated p38 MAPKs through the production of singlet oxygen. Inhibition of p38 MAPKs with chemical inhibitors or siRNA enhances HY-PDT-induced apoptosis of the HK-1 NPC cells.     

Inhibitory effects of enalaprilat on rat cardiac fibroblast proliferation via ROS/P38MAPK/TGF-β1 signaling pathway

Enalaprilat (Ena.), an angiotensin II (Ang II) converting enzyme inhibitor (ACEI), can produce some therapeutic effects on hypertension, ventricular hypertrophy and myocardial remodeling in clinic, but its precise mechanism, especially its signaling pathways remain elusive. In this study, cardiac fibroblasts (CFb) was isolated by the trypsin digestion method; a BrdU proliferation assay was adopted to determine cell proliferation; an immunofluorescence assay was used to measure intracellular reactive oxygen species (ROS); immunocytochemistry staining and Western blotting assay were used to detect phosphorylated p38 mitogen activated protein kinase (p-p38MAPK) and transforming growth factor-β(1) (TGF-β(1)) protein expression, respectively. The results showed that Ang II (10(-7) M) stimulated the cardiac fibroblast proliferation which was inhibited by NAC (an antioxidant), SB203580 (a p38MAPK inhibitor) or enalaprilat; Ang II caused an burst of intracellular ROS level within thirty minutes, an increase in p-p38MAPK (3.6-fold of that in the control group), as well as an elevation of TGF-β(1) meantime; NAC, an antioxidant, and enalaprilat treatment attenuated cardiac fibroblast proliferation induced by Ang II and decreased ROS and p-p38MAPK protein levels in rat cardiac fibroblast; SB203580 lowered TGF-β(1) protein expression in rats' CFb in a dose-dependent manner. It could be concluded that enalaprilat can inhibit the cardiac fibroblast proliferation induced by Ang II via blocking ROS/P38MAPK/TGF-β(1) signaling pathways and the study provides a theoretical proof for the application of ACEIs in treating myocardial fibrosis and discovering the primary mechanism through which ACEIs inhibit CFb proliferation.