Selective dysfunction of p53 for mitochondrial biogenesis induces cellular proliferation in bronchial smooth muscle from asthmatic patients.

BACKGROUND: Increase of bronchial smooth muscle (BSM) mass is a crucial feature of asthma remodeling. The mechanisms of such an increased BSM mass are complex but involve enhanced mitochondrial biogenesis, leading to increased proliferation of BSM cells in asthmatic patients. The major tumor suppressor protein p53 is a key cell regulator involved in cell proliferation and has also been implicated in mitochondrial biogenesis. However, the role of p53 in BSM cell proliferation and mitochondrial biogenesis has not been investigated thus far. OBJECTIVE: We sought to evaluate the role of p53 in proliferation of BSM cells in asthmatic patients and mitochondrial biogenesis. METHODS: The expression of p53 was assessed both in vitro by using flow cytometry and Western blotting and ex vivo by using RT-PCR after laser microdissection. The role of p53 was assessed with small hairpin RNA lentivirus in both asthmatic patients and control subjects with BSM cell proliferation by using 5-bromo-2'-deoxyuridine and cell counting and in the expression of p21, BCL2-associated X protein, mitochondrial transcription factor A (TFAM), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). RESULTS: Twenty-nine patients with moderate-to-severe asthma and 26 control subjects were enrolled in the study. p53 expression was increased in BSM from asthmatic patients both ex vivo and in vitro, with a decreased interaction with mouse double minute 2 homolog (Mdm2) and an increased phosphorylation of serine 20. p53 did not inhibit the transcription of both TFAM and PGC-1α in BSM cells from asthmatic patients. As a consequence, p53 is unable to slow the increased mitochondrial biogenesis and hence the subsequent increased proliferation of BSM cells in asthmatic patients. CONCLUSION: This study suggests that p53 might act as a new potential therapeutic target against BSM remodeling in asthmatic patients.

Effect of hypoxia on TRPV1 and TRPV4 channels in rat pulmonary arterial smooth muscle cells.

Transient receptor potential (TRP) channels of the vanilloid subfamily, mainly TRPV1 and TRPV4, are expressed in pulmonary artery smooth muscle cells (PASMC) and implicated in the remodeling of pulmonary artery, a landmark of pulmonary hypertension (PH). Among a variety of PH subtypes, PH of group 3 are mostly related to a prolonged hypoxia exposure occurring in a variety of chronic lung diseases. In the present study, we thus investigated the role of hypoxia on TRPV1 and TRPV4 channels independently of the increased pulmonary arterial pressure that occurs during PH. We isolated PASMC from normoxic rat and cultured these cells under in vitro hypoxia. Using microspectrofluorimetry and the patch-clamp technique, we showed that hypoxia (1 % O2 for 48 h) significantly increased stretch- and TRPV4-induced calcium responses. qRT-PCR, Western blotting, and immunostaining experiments revealed that the expression of TRPV1 and TRPV4 was not enhanced under hypoxic conditions, but we observed a membrane translocation of TRPV1. Furthermore, hypoxia induced a reorganization of the F-actin cytoskeleton, the tubulin, and intermediate filament networks (immunostaining experiments), associated with an enhanced TRPV1- and TRPV4-induced migratory response (wound-healing assay). Finally, as assessed by immunostaining, exposure to in vitro hypoxia elicited a significant increase in NFATc4 nuclear localization. Cyclosporin A and BAPTA-AM inhibited NFATc4 translocation, indicating the activation of the Ca(2+)/calcineurin/NFAT pathway. In conclusion, these data point out the effect of hypoxia on TRPV1 and TRPV4 channels in rat PASMC, suggesting that these channels can act as direct signal transducers in the pathophysiology of PH.

Involvement of TRPV1 and TRPV4 channels in migration of rat pulmonary arterial smooth muscle cells.

Pulmonary hypertension, the main disease of the pulmonary circulation, is characterized by an increase in pulmonary vascular resistance, involving proliferation and migration of pulmonary arterial smooth muscle cells (PASMC). However, cellular and molecular mechanisms underlying these phenomena remain to be identified. In the present study, we thus investigated in rat intrapulmonary arteries (1) the expression and the functional activity of TRPV1 and TRPV4, (2) the PASMC migration triggered by these TRPV channels, and (3) the associated reorganization of the cytoskeleton. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated expression of TRPV1 and TRPV4 mRNA in rat intrapulmonary arteries. These results were confirmed at the protein level by western blot. Using microspectrofluorimetry (indo-1), we show that capsaicin and 4α-phorbol-12,13-didecanoate (4α-PDD), selective agonists of TRPV1 and TRPV4, respectively, increased the intracellular calcium concentration of PASMC. Furthermore, stimulation of TRPV1 and TRPV4 induced PASMC migratory responses, as assessed by two different methods (a modified Boyden chamber assay and a wound-healing migration assay). This response cannot seem to be attributed to a proliferative effect as assessed by BrdU and Wst-1 colorimetric methods. Capsaicin- and 4α-PDD-induced calcium and migratory responses were inhibited by the selective TRPV1 and TRPV4 blockers, capsazepine and HC067047, respectively. Finally, as assessed by immunostaining, these TRPV-induced migratory responses were associated with reorganization of the F-actin cytoskeleton and the tubulin and intermediate filament networks. In conclusion, these data point out, for the first time, the implication of TRPV1 and TRPV4 in rat PASMC migration, suggesting the implication of these TRPV channels in the physiopathology of pulmonary hypertension.

Signaling Pathways Linked to Serotonin-Induced Superoxide Anion Production: A Physiological Role for Mitochondria in Pulmonary Arteries.

Serotonin (5-HT) is a potent vasoconstrictor agonist and contributes to several vascular diseases including systemic or pulmonary hypertension and atherosclerosis. Although superoxide anion ([Formula: see text]) is commonly associated to cellular damages due to [Formula: see text] overproduction, we previously demonstrated that, in physiological conditions, [Formula: see text] also participates to the 5-HT contraction in intrapulmonary arteries (IPA). Here, we focused on the signaling pathways leading to [Formula: see text] production in response to 5-HT in rat IPA. Using electron paramagnetic resonance on rat IPA, we showed that 5-HT (100 µM)-induced [Formula: see text] production was inhibited by ketanserin (1 µM-an inhibitor of the 5-HT2 receptor), absence of extracellular calcium, two blockers of voltage-independent calcium permeable channels (RHC80267 50 µM and LOE-908 10 µM) and a blocker of the mitochondrial complex I (rotenone-100 nM). Depletion of calcium from the sarcoplasmic reticulum or nicardipine (1 µM-an inhibitor of the L-type voltage-dependent calcium channel) had no effect on the 5-HT-induced [Formula: see text] production. [Formula: see text] levels were also increased by α-methyl-5-HT (10 µM-a 5-HT2 receptors agonist) whereas GR127935 (1 µM-an antagonist of the 5-HT1B/D receptor) and citalopram (1 µM-a 5-HT transporter inhibitor) had no effect on the 5-HT-induced [Formula: see text] production. Peroxynitrites were increased in response to 5-HT (100 µM). In isolated pulmonary arterial smooth muscle cells loaded with rhod-2 or mitosox probes, we respectively showed that 5-HT increased both mitochondrial calcium and [Formula: see text] levels, which were both abrogated in absence of extracellular calcium. Mitochondrial [Formula: see text] levels were also abolished in the presence of rotenone (100 nM). In pulmonary arterial smooth muscle cells loaded with TMRM, we showed that 5-HT transiently depolarized the mitochondrial membrane whereas in the absence of extracellular calcium the mitochondrial membrane depolarisation was delayed and sustained in response to 5-HT. 5-HT decreased the mitochondrial respiratory rate measured with a Clark oxygen electrode. Altogether, in physiological conditions, 5-HT acts on 5-HT2 receptors and induces an [Formula: see text] production dependent on extracellular calcium and mitochondria.

Identification and characterization of two dipeptidyl-peptidase III isoforms in Drosophila melanogaster.

Dipeptidyl-peptidase III (DPP III) hydrolyses small peptides with a broad substrate specificity. It is thought to be involved in a major degradation pathway of the insect neuropeptide proctolin. We report the purification and characterization of a soluble DPP III from 40 g Drosophila melanogaster. Western blot analysis with anti-(DPP III) serum revealed the purification of two proteins of molecular mass 89 and 82 kDa. MS/MS analysis of these proteins resulted in the sequencing of 45 and 41 peptide fragments, respectively, confirming approximately 60% of both annotated D. melanogaster DPP III isoforms (CG7415-PC and CG7415-PB) predicted at 89 and 82 kDa. Sequencing also revealed the specific catalytic domain HELLGH in both isoforms, indicating that they are both effective in degrading small peptides. In addition, with a probe specific for D. melanogaster DPP III, northern blot analysis of fruit fly total RNA showed two transcripts at approximately 2.6 and 2.3 kb, consistent with the translation of 89-kDa and 82-kDa DPP III proteins. Moreover, the purified enzyme hydrolyzed the insect neuropeptide proctolin (Km approximately 4 microm) at the second N-terminal peptide bound, and was inhibited by the specific DPP III inhibitor tynorphin. Finally, anti-(DPP III) immunoreactivity was observed in the central nervous system of D. melanogaster larva, supporting a functional role for DPP III in proctolin degradation. This study shows that DPP III is in actuality synthesized in D. melanogaster as 89-kDa and 82-kDa isoforms, representing two native proteins translated from two alternative mRNA transcripts.

Protease activated receptor-2 expression and function in asthmatic bronchial smooth muscle.

Asthmatic bronchial smooth muscle (BSM) is characterized by structural remodeling associated with mast cell infiltration displaying features of chronic degranulation. Mast cell-derived tryptase can activate protease activated receptor type-2 (PAR-2) of BSM cells. The aims of the present study were (i) to evaluate the expression of PAR-2 in both asthmatic and non asthmatic BSM cells and, (ii) to analyze the effect of prolonged stimulation of PAR-2 in asthmatic BSM cells on cell signaling and proliferation. BSM cells were obtained from both 33 control subjects and 22 asthmatic patients. PAR-2 expression was assessed by flow cytometry, western blot and quantitative RT-PCR. Calcium response, transduction pathways and proliferation were evaluated before and following PAR-2 stimulation by SLIGKV-NH2 or trypsin for 1 to 3 days. Asthmatic BSM cells expressed higher basal levels of functional PAR-2 compared to controls in terms of mRNA, protein expression and calcium response. When PAR-2 expression was increased by means of lentivirus in control BSM cells to a level similar to that of asthmatic cells, PAR-2-induced calcium response was then similar in both types of cell. However, repeated PAR-2 stimulations increased the proliferation of asthmatic BSM cells but not that of control BSM cells even following lentiviral over-expression of PAR-2. Such an increased proliferation was related to an increased phosphorylation of ERK in asthmatic BSM cells. In conclusion, we have demonstrated that asthmatic BSM cells express increased baseline levels of functional PAR-2. This higher basal level of PAR-2 accounts for the increased calcium response to PAR-2 stimulation, whereas the increased proliferation to repeated PAR-2 stimulation is related to increased ERK phosphorylation.

Liver myofibroblasts activate protein C and respond to activated protein C.

AIM: To study the protein C activation system in human liver myofibroblasts, and the effects of activated protein C (APC) on these cells. METHODS: Human liver myofibroblasts were obtained by outgrowth. Expression of protease activated receptor 1 (PAR-1), endothelial protein C receptor (EPCR) and thrombomodulin (TM) was analyzed by flow cytometry. Extracellular signal-regulated kinase (ERK)1/2 activation was assessed by Western blotting using anti-phospho-ERK antibodies. Collagen synthesis was studied with real-time reverse transcription-polymerase chain reaction (RT-PCR). Activation of protein C was studied by incubating liver myofibroblasts with zymogen protein C in the presence of thrombin and detecting the generation of APC with a colorimetric assay using a peptide substrate. RESULTS: Primary cultures of human liver myofibroblasts expressed EPCR on their surface, together with PAR-1 and TM. This receptor system was functional since exposure of myofibroblasts to APC induced ERK1/2 phosphorylation in a dose- and time-dependent manner. Furthermore, APC significantly upregulated the expression of collagen mRNA, as shown by real-time RT-PCR. Collagen upregulation was controlled through the ERK pathway as it was inhibited when using the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor PD98059. Finally, using a cell-based colorimetric assay, we showed that intact myofibroblasts converted protein C into APC in the presence of thrombin. CONCLUSION: These data suggest that APC is a new modulator of liver myofibroblast activity and contributes to the pathophysiology of chronic liver diseases.

Protease-activated receptor 1 knockout reduces experimentally induced liver fibrosis.

Thrombin inhibition protects against liver fibrosis. However, it is not known whether the thrombin profibrogenic effect is due to effects on blood coagulation or to signaling via protease-activated receptors (PARs). We took advantage of the lack of blood coagulation defects in PAR-1-knockout mice. Acute carbon tetrachloride (CCl(4)) toxicity was similar in wild-type (WT), PAR-1(-/-), and PAR-1(+/-) mice as judged by aminotransferase levels, area of liver necrosis, and liver peroxidation measured by Fourier-transformed infrared spectroscopy. Fifteen mice/group received CCl(4) or its solvent for 6 wk (300 microl/kg, 3 times a week). Fibrosis area was increased 10-fold by CCl(4) treatment in WT mice. PAR-1 deficiency protected against fibrosis, with 36% and 56% decrease in PAR-1(+/-) and PAR-1(-/-) mice, respectively (P < 0.001). Similar results were obtained for area of activated fibrogenic cells (64% and 79% decrease in PAR-1(+/-) and PAR-1(-/-) mice, respectively, P < 0.001). These findings were corroborated by measurements of type I collagen, matrix metalloproteinase-2, and PDGF-beta receptor mRNA levels. There was also a significant decrease in T lymphocyte infiltration in PAR-1-deficient mice. Altogether, these results suggest that thrombin profibrogenic effects are independent of effects on blood coagulation and are instead due to direct effects on fibrogenic cells and possibly on T lymphocytes.

Thrombin inhibits migration of human hepatic myofibroblasts.

Several lines of data recently pointed out a role of the serine proteinase thrombin in liver fibrogenesis, but its mechanism of action is unknown. The aim of this study was to evaluate the effect of thrombin on the migration of human liver myofibroblasts. We show here that thrombin inhibits both basal migration and platelet-derived growth factor (PDGF)-BB-induced migration of myofibroblasts. By using a thrombin antagonist, a protease-activated receptor (PAR)-1 mimetic peptide, and a PAR-1 antibody, we show that this effect is dependent on the catalytic activity of thrombin and on PAR-1 activation. Thrombin's effect on basal migration was dependent on cyclooxygenase 2 (COX-2) activation because it was blocked by the COX-2 inhibitors NS-398 and nimesulide, and pharmacological studies showed that it was relayed through prostaglandin E(2) and its EP(2) receptor. On the other hand, thrombin-induced inhibition of PDGF-BB-induced migration was not dependent on COX-2. We show that thrombin inhibits PDGF-induced Akt-1 phosphorylation. This effect was consecutive to inhibition of PDGF-beta receptor activation through active dephosphorylation. Thus thrombin, through two distinct mechanisms, inhibits both basal- and PDGF-BB-induced migration of human hepatic liver myofibroblasts. The fine tuning of myofibroblast migration may be one of the mechanisms used by thrombin to regulate liver fibrogenesis.