Mammalian Target of Rapamycin (mTOR) Induces Proliferation and De-Differentiation Responses to Three Coordinate Pathophysiologic Stimuli (Mechanical Strain, Hypoxia, and Extracellular Matrix Remodeling) in Rat Bladder Smooth Muscle
2009; Elsevier BV; Volume: 176; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2010.080834
ISSN1525-2191
AutoresKaren Aitken, Cornelia Tölg, Trupti Panchal, Bruno Leslie, Jeffery Yu, Mohamed S. Elkelini, N. Sabha, Derrick Tse, Armando J. Lorenzo, Magdy Hassouna, Darius Bägli,
Tópico(s)Pulmonary Hypertension Research and Treatments
ResumoMaladaptive bladder muscle overgrowth and de-differentiation in human bladder obstructive conditions is instigated by coordinate responses to three stimuli: mechanical strain, tissue hypoxia, and extracellular matrix remodeling.1Mattiasson A Uvelius B Changes in contractile properties in hypertrophic rat urinary bladder.J Urol. 1982; 128: 1340-1342PubMed Google Scholar, 2Becker A Baum M Obstructive uropathy.Early Hum Dev. 2006; 82: 15-22Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Pathway analysis of genes induced by obstructive models of injury in bladder smooth muscle cells (BSMCs) identified a mammalian target of rapamycin (mTOR)-specific inhibitor as a potential pharmacological inhibitor. Strain-induced mTOR-specific S6K activation segregated differently from ERK1/2 activation in intact bladder ex vivo. Though rapamycin's antiproliferative effects in vascular smooth muscle cells are well known, its effects on BSMCs were previously unknown. Rapamycin significantly inhibited proliferation of BSMCs in response to mechanical strain, hypoxia, and denatured collagen. Rapamycin inhibited S6K at mTOR-sensitive phosphorylation sites in response to strain and hypoxia. Rapamycin also supported smooth muscle actin expression in response to strain or hypoxia-induced de-differentiation. Importantly, strain plus hypoxia synergistically augmented mTOR-dependent S6K activation, Mmp7 expression and proliferation. Forced expression of wild-type and constitutively active S6K resulted in loss of smooth muscle actin expression. Decreased smooth muscle actin, increased Mmp7 levels and mTOR pathway activation during in vivo partial bladder obstruction paralleled our in vitro studies. These results point to a coordinate role for mTOR in BSMCs responses to the three stimuli and a potential new therapeutic target for myopathic bladder disease. Maladaptive bladder muscle overgrowth and de-differentiation in human bladder obstructive conditions is instigated by coordinate responses to three stimuli: mechanical strain, tissue hypoxia, and extracellular matrix remodeling.1Mattiasson A Uvelius B Changes in contractile properties in hypertrophic rat urinary bladder.J Urol. 1982; 128: 1340-1342PubMed Google Scholar, 2Becker A Baum M Obstructive uropathy.Early Hum Dev. 2006; 82: 15-22Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Pathway analysis of genes induced by obstructive models of injury in bladder smooth muscle cells (BSMCs) identified a mammalian target of rapamycin (mTOR)-specific inhibitor as a potential pharmacological inhibitor. Strain-induced mTOR-specific S6K activation segregated differently from ERK1/2 activation in intact bladder ex vivo. Though rapamycin's antiproliferative effects in vascular smooth muscle cells are well known, its effects on BSMCs were previously unknown. Rapamycin significantly inhibited proliferation of BSMCs in response to mechanical strain, hypoxia, and denatured collagen. Rapamycin inhibited S6K at mTOR-sensitive phosphorylation sites in response to strain and hypoxia. Rapamycin also supported smooth muscle actin expression in response to strain or hypoxia-induced de-differentiation. Importantly, strain plus hypoxia synergistically augmented mTOR-dependent S6K activation, Mmp7 expression and proliferation. Forced expression of wild-type and constitutively active S6K resulted in loss of smooth muscle actin expression. Decreased smooth muscle actin, increased Mmp7 levels and mTOR pathway activation during in vivo partial bladder obstruction paralleled our in vitro studies. These results point to a coordinate role for mTOR in BSMCs responses to the three stimuli and a potential new therapeutic target for myopathic bladder disease. Conditions that impede bladder emptying, incite chronic distension, or overstimulate neuromuscular activity in the bladder wall cause high pressure and strain, leading to hypoxia, extracellular matrix (ECM) remodeling, and smooth muscle overgrowth.1Mattiasson A Uvelius B Changes in contractile properties in hypertrophic rat urinary bladder.J Urol. 1982; 128: 1340-1342PubMed Google Scholar, 2Becker A Baum M Obstructive uropathy.Early Hum Dev. 2006; 82: 15-22Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 3Austin JC Chacko SK DiSanto M Canning DA Zderic SA A male murine model of partial bladder outlet obstruction reveals changes in detrusor morphology, contractility and Myosin isoform expression.J Urol. 2004; 172: 1524-1528Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 4Buttyan R Chen MW Levin RM Animal models of bladder outlet obstruction and molecular insights into the basis for the development of bladder dysfunction.Eur Urol. 1997; 32 Suppl 1: 32-39PubMed Google Scholar, 5Johansson R Persson K Phenotypic modulation of cultured bladder smooth muscle cells and the expression of inducible nitric oxide synthase.Am J Physiol Regul Integr Comp Physiol. 2004; 286: R642-R648Crossref PubMed Scopus (8) Google Scholar, 6Krishna A Lal P Gupta A Madan U Posterior urethral valves after infancy-urodynamic consequences.Ped Surg Int. 1998; 13: 504-507Crossref PubMed Scopus (7) Google Scholar, 7Amaro JL Balasteghin KT Padovani CR Montenegro R Structural alterations of the bladder induced by detrusor instability. Experimental study in rabbits.Int Braz J Urol. 2005; 31: 579-585Crossref PubMed Scopus (8) Google Scholar, 8Greenland JE Hvistendahl JJ Andersen H Jorgensen TM McMurray G Cortina-Borja M Brading AF Frøkiaer J The effect of bladder outlet obstruction on tissue oxygen tension and blood flow in the pig bladder.BJU Int. 2000; 85: 1109-1114Crossref PubMed Google Scholar Other diseases, such as atherosclerosis, have significant consequences triggering “phenotypic switching” of smooth muscle cells (SMCs) from contractile to proliferative, hypertrophic, or synthetic phenotypes. In the bladder, SMC phenotypic alterations resulting from obstruction1Mattiasson A Uvelius B Changes in contractile properties in hypertrophic rat urinary bladder.J Urol. 1982; 128: 1340-1342PubMed Google Scholar, 2Becker A Baum M Obstructive uropathy.Early Hum Dev. 2006; 82: 15-22Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 3Austin JC Chacko SK DiSanto M Canning DA Zderic SA A male murine model of partial bladder outlet obstruction reveals changes in detrusor morphology, contractility and Myosin isoform expression.J Urol. 2004; 172: 1524-1528Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 4Buttyan R Chen MW Levin RM Animal models of bladder outlet obstruction and molecular insights into the basis for the development of bladder dysfunction.Eur Urol. 1997; 32 Suppl 1: 32-39PubMed Google Scholar may lead to dysfunctional micturition, and bladder decompensation. Distension or wall tension is the stimulus initiating signaling or mechanotransduction in the bladder wall, and can lead to intramural and microvascular compression. Transmural tension and compression of the intramural microvasculature creates tissue hypoxia during bladder obstruction.4Buttyan R Chen MW Levin RM Animal models of bladder outlet obstruction and molecular insights into the basis for the development of bladder dysfunction.Eur Urol. 1997; 32 Suppl 1: 32-39PubMed Google Scholar, 8Greenland JE Hvistendahl JJ Andersen H Jorgensen TM McMurray G Cortina-Borja M Brading AF Frøkiaer J The effect of bladder outlet obstruction on tissue oxygen tension and blood flow in the pig bladder.BJU Int. 2000; 85: 1109-1114Crossref PubMed Google Scholar Both hypoxic and distensive stimuli in bladder smooth muscle cells (BSMCs) can lead to matrix metalloproteinase (MMP) activation9Sabha N Aitken K Lorenzo AJ Szybowska M Jairath A Bägli DJ Matrix metalloproteinase-7 and epidermal growth factor receptor mediate hypoxia-induced extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activation and subsequent proliferation in bladder smooth muscle cells.In Vitro Cell Dev Biol Animal. 2006; 42: 124-133Crossref PubMed Scopus (14) Google Scholar, 10Aitken KJ Block G Lorenzo A Herz D Sabha N Dessouki O Fung F Szybowska M Craig L Bägli DJ Mechanotransduction of extracellular signal-regulated kinases 1 and 2 mitogen-activated protein kinase activity in smooth muscle is dependent on the extracellular matrix and regulated by matrix metalloproteinases.Am J Pathol. 2006; 169: 459-470Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 11Herz DB Aitken K Bägli DJ Collagen directly stimulates bladder smooth muscle cell growth in vitro: regulation by extracellular regulated mitogen activated protein kinase.J Urol. 2003; 170: 2072-2076Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar and matrix remodeling. These remodeling events can expose cryptic epitopes within native matrix elements driving further BSMC growth, which is often self-perpetuating.10Aitken KJ Block G Lorenzo A Herz D Sabha N Dessouki O Fung F Szybowska M Craig L Bägli DJ Mechanotransduction of extracellular signal-regulated kinases 1 and 2 mitogen-activated protein kinase activity in smooth muscle is dependent on the extracellular matrix and regulated by matrix metalloproteinases.Am J Pathol. 2006; 169: 459-470Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 11Herz DB Aitken K Bägli DJ Collagen directly stimulates bladder smooth muscle cell growth in vitro: regulation by extracellular regulated mitogen activated protein kinase.J Urol. 2003; 170: 2072-2076Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar Despite the widespread and well-recognized clinical sequelae of obstructive uropathies, the signaling mechanisms driving excessive proliferation and phenotypic switching of BSMCs have not been adequately addressed. This knowledge gap has further impeded the development of new pharmacotherapy for obstructive uropathy. Numerous studies have illustrated the cell-cycle kinases and mitogen activated protein kinases involved in accelerated vascular SMC growth,12Watson MH Venance SL Pang SC Mak AS Smooth muscle cell proliferation. Expression and kinase activities of p34cdc2 and mitogen-activated protein kinase homologues.Circ Res. 1993; 73: 109-117Crossref PubMed Scopus (35) Google Scholar but in the bladder, the signaling pathways and the critical physiological stimuli driving them are only beginning to be understood.13Adam RM Recent insights into the cell biology of bladder smooth muscle.Nephron Exp Nephrol. 2006; 102: e1-e7Crossref PubMed Scopus (10) Google Scholar Bioinformatics analysis of previously identified genes involved in three models of BSMC injury suggested that rapamycin could exert an inhibitory affect on a major associated gene network. Rapamycin, a macrolide antibiotic, and specific inhibitor of the mammalian target of rapamycin (mTOR, or FRAP1), has been used widely to inhibit development of transplant arteriosclerosis and arterial neointimal thickening of vascular smooth muscle following mechanical and alloimmune injury.14Gregory CR Pratt RE Huie P Shorthouse R Dzau VJ Billingham ME Morris RE Effects of treatment with cyclosporine, FK 506, rapamycin, mycophenolic acid, or deoxyspergualin on vascular muscle proliferation in vitro and in vivo.Transplantation Proc. 1993; 25: 770-771PubMed Google Scholar Further, this Food and Drug Administration-approved drug15Thompson CA First drug-eluting coronary stent approved.AJHP. 2003; 60: 1210-1212Google Scholar has been effective in treating advanced renal cell carcinoma, among other cancer types16Faivre S, Kroemer G, Raymond E: Current development of mTOR inhibitors as anticancer agents. Nat Rev Drug Disc 5:671–688Google Scholar and strongly prevents organ rejection in renal and other transplants. mTOR plays a pivotal role in cell cycle progression and differentiation in vascular smooth muscle cells via orchestration of kinase activity and protein translation.17Sakakibara K Liu B Hollenbeck S Kent KC Rapamycin inhibits fibronectin-induced migration of the human arterial smooth muscle line (E47) through the mammalian target of rapamycin.Am J Physiol Heart Circ Physiol. 2005; 288: H2861-H2868Crossref PubMed Scopus (30) Google Scholar, 18Fingar DC Richardson CJ Tee AR Cheatham L Tsou C Blenis J mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E.Mol Cell Biol. 2004; 24: 200-216Crossref PubMed Scopus (709) Google Scholar This signaling pathway directs translation of 5′CAP and 5′TOP mRNAs through phosphorylation of S6 kinases18Fingar DC Richardson CJ Tee AR Cheatham L Tsou C Blenis J mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E.Mol Cell Biol. 2004; 24: 200-216Crossref PubMed Scopus (709) Google Scholar and EIF4E,18Fingar DC Richardson CJ Tee AR Cheatham L Tsou C Blenis J mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E.Mol Cell Biol. 2004; 24: 200-216Crossref PubMed Scopus (709) Google Scholar respectively, augmenting cell size, as well as cell number. The inhibitor of this pathway, rapamycin, was able to modulate BSMC phenotype under the mitogenic conditions of mechanical strain and hypoxia (both together and separately), and denatured matrix, three defining stimuli of the obstructive uropathic microenvironment in vivo. Also, mTOR and its inhibitor rapamycin were able to alter expression of smooth muscle actin (SMA), a well-studied early differentiation marker for SMC, in three different physiological models of BSMC injury. Furthermore, we assessed involvement of downstream effectors of mTOR, including S6K1, in differentiation of BSMC. Ingenuity Pathways Analysis (IPA, Ingenuity Systems, Inc.A Redwood City, CA) was used to identify highly associated networks of genes and pathways involved in BSMC strain and hypoxia injury. Using genes from previous work9Sabha N Aitken K Lorenzo AJ Szybowska M Jairath A Bägli DJ Matrix metalloproteinase-7 and epidermal growth factor receptor mediate hypoxia-induced extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activation and subsequent proliferation in bladder smooth muscle cells.In Vitro Cell Dev Biol Animal. 2006; 42: 124-133Crossref PubMed Scopus (14) Google Scholar, 10Aitken KJ Block G Lorenzo A Herz D Sabha N Dessouki O Fung F Szybowska M Craig L Bägli DJ Mechanotransduction of extracellular signal-regulated kinases 1 and 2 mitogen-activated protein kinase activity in smooth muscle is dependent on the extracellular matrix and regulated by matrix metalloproteinases.Am J Pathol. 2006; 169: 459-470Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 19Halachmi H Sarel S Aitken A Karen K Szybowska S Marta M Sabha S Nesrin N Dessouki D Shariff S Lorenzo L Armando A Tse T Derrick D Bagli B Darius D Role of signal transducer and activator of transcription 3 (STAT3) in stretch injury to bladder smooth muscle cells.Cell Tissue Res. 2006; 326: 149-158Crossref PubMed Scopus (23) Google Scholar, 20Elkelini MS Aitken K Bagli DJ Hassouna MM Effects of doxycycline on voiding behaviour of rats with bladder outlet obstruction.BJU Int. 2009; 103: 537-540Crossref PubMed Scopus (7) Google Scholar, 21Adam RM Eaton SH Estrada C Nimgaonkar A Shih S-C Smith LEH Kohane IS Bägli D Freeman MR Mechanical stretch is a highly selective regulator of gene expression in human bladder smooth muscle cells.Physiol Genomics. 2004; 20: 36-44Crossref PubMed Scopus (84) Google Scholar and the known association of muscarinic receptors with obstructive uropathy, focus genes (listed in Table 1) were mapped to gene identifiers in the IPA knowledge base and overlaid on a molecular network curated by IPA. Gene identifiers were mapped to networks based on their known connectivity and given a score based on the number of focus gene identifiers found in the networks. This score is not a significance score, but simply ranks the networks according to their relevance to the focus gene identifiers. The two most significant networks identified from this analysis were queried for potential chemical or biological inhibitors of these pathways by examining the genes associated with the networks for chemical biological and inhibitors listed in the gene database on IPA. Inhibitors identified were screened for practical applicability based on clinical availability and toxicity. The data were also mapped to canonical pathways and significance of these associations determined by both a ratio of the number of focus gene identifiers mapping to the canonical pathway versus the total number of gene identifiers mapping to the canonical pathway and a one-sided Fisher's exact test was used to uncover pathways of genes with higher odds ratios of containing our focus genes. Some genes identified by the gene networks were in fact groups or complexes of genes, for example “MMP,” “MEK,” “ERK,” or Gαi, as the data curated by IPA in some cases is not specific to one gene but a group of genes.Table 1List of Focus Genes from Figure 1AGeneBladder SMC stimulusName and aliasGeneral function in SMCAKT1PDGF, strain and pressure induced proliferationV-akt murine thymoma viral oncogene homolog 1ProliferationAKT2PDGF, strain and pressure induced proliferationV-akt murine thymoma viral oncogene homolog 2DifferentiationAKT3PDGF, strain and pressure induced proliferationV-akt murine thymoma viral oncogene homolog 3ProliferationBMP2StrainBone-morphogenic protein-2Anti-proliferative, apoptosisBMPR2StrainBone-morphogenic protein receptor-2Growth arrest, differentiationCHRM2ObstructionMuscarinic acetylcholine receptor 2Cholinergic contractionCHRM3ObstructionMuscarinic acetylcholine receptor 3Cholinergic contractionCHRM4ObstructionMuscarinic acetylcholine receptor 4Cholinergic contractionCOL3A1Obstruction, strainCollagen type III (alpha 1)Matrix scaffoldF2RL1StrainCoagulation factor II (thrombin) receptor-like 1, protease activated receptor 2 (PAR2′)Migration, neuroactive ligand-receptor interactionHBEGFStrainHeparin-binding EGF-like growth factor (HB-EGF)Proliferation, hypertrophy remodelingLIFStrainLeukemia inhibitory factorNOS inductionMAPK1Strain + hypoxiaERK2, mitogen-activated protein kinase 1Differentiation, proliferation, apoptosis, migrationMAPK14Strainp38, mitogen-activated protein kinase 14Migration (MMP expression), neointimal growth, apoptosisMAPK3Strain + hypoxiaERK1, mitogen-activated protein kinase 3Differentiation, proliferation, apoptosis, migrationMAPK8StrainJNK1, mitogen-activated protein kinase 8ProliferationMMP2StrainMatrix metalloproteinase 2Migration, proliferationMMP7Strain + hypoxiaMatrix metalloproteinase 7ProliferationMMP9StrainMatrix metalloproteinase 9Migration, proliferationPTGS2StrainProstaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)ProliferationSTAT3Strain + obstructionSignal transducers and activator of transcription 3Proliferation, angiogenesis, apoptosis (IFNγ-induced) Open table in a new tab Bladders from neonatal Sprague-Dawley rats (2 to 5 days old) were isolated and SMCs isolated as previously described.8Greenland JE Hvistendahl JJ Andersen H Jorgensen TM McMurray G Cortina-Borja M Brading AF Frøkiaer J The effect of bladder outlet obstruction on tissue oxygen tension and blood flow in the pig bladder.BJU Int. 2000; 85: 1109-1114Crossref PubMed Google Scholar Eagle's minimum essential medium (EMEM; Multicell Techologies, Woonsocket, Rhode Island) containing 10% fetal calf serum (Invitrogen Carlsbad, CA) and antibiotic/antimycotic (Multicell) was used to culture cells at 37°C in 95%O2/5%CO29Sabha N Aitken K Lorenzo AJ Szybowska M Jairath A Bägli DJ Matrix metalloproteinase-7 and epidermal growth factor receptor mediate hypoxia-induced extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activation and subsequent proliferation in bladder smooth muscle cells.In Vitro Cell Dev Biol Animal. 2006; 42: 124-133Crossref PubMed Scopus (14) Google Scholar as described previously. Passages 1 and 2 were used for experiments in this study. To passage and plate cells, BSMC were incubated in 0.25% trypsin, 0.053 mmol/L EDTA (Multicell) briefly, washed in PBS, and re-suspended at 5 × 104 cells/ml. For proliferation assays, 0.5 × 105 cells were seeded into 6 well tissue culture plates or BioFlex plates (Flexcell International, Inc.). Before all experiments, cells were serum-deprived using starvation media (EMEM without serum) for 48 hours to synchronize BSMCs to G0. Type I bovine collagen (Elastin Products Company, Owensville, Missouri) was gelated by neutralizing the collagen solution in 0.1 mol/L NaOH in 1× PBS (MultiCell) at 37°C. Collagen was denatured by boiling for 30 minutes, then neutralized in 0.1mol/L NaOH as described.9Sabha N Aitken K Lorenzo AJ Szybowska M Jairath A Bägli DJ Matrix metalloproteinase-7 and epidermal growth factor receptor mediate hypoxia-induced extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activation and subsequent proliferation in bladder smooth muscle cells.In Vitro Cell Dev Biol Animal. 2006; 42: 124-133Crossref PubMed Scopus (14) Google Scholar For denatured collagen (DNC) gels, native collagen (NC) was layered with an equivalent volume of DNC overnight before washing. Both DNC and NC gels were washed in EMEM before plating cells. BSMC were serum-starved for 48 hours before plating at a density of 2 × 104 cells/ml for 3 hours before addition of any pharmacological agent. Cells were incubated for 48 hours at 37°C, 5% CO2. Cells were mechanically strained on a vacuum modulated device (Flexcell 4000, Flexcell International Corporation), 5 × 104 cells/ml were plated onto Bioflex Collagen I strain plates. At 50% confluency, cells were serum-starved for 48 hours to arrest cells at the G0. All strain experiments were conducted using a static pattern with an initial ramping of 2% and 4% elongation for 1 hour each, then 5% elongation for a total of 16 or 18 hours strain.10Aitken KJ Block G Lorenzo A Herz D Sabha N Dessouki O Fung F Szybowska M Craig L Bägli DJ Mechanotransduction of extracellular signal-regulated kinases 1 and 2 mitogen-activated protein kinase activity in smooth muscle is dependent on the extracellular matrix and regulated by matrix metalloproteinases.Am J Pathol. 2006; 169: 459-470Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar This static patterning is more reminiscent of slow bladder filling and chronic distension, rather than rapidly cyclic or oscillating straining patterns more appropriate for vascular SMC.10Aitken KJ Block G Lorenzo A Herz D Sabha N Dessouki O Fung F Szybowska M Craig L Bägli DJ Mechanotransduction of extracellular signal-regulated kinases 1 and 2 mitogen-activated protein kinase activity in smooth muscle is dependent on the extracellular matrix and regulated by matrix metalloproteinases.Am J Pathol. 2006; 169: 459-470Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar To generate a controlled, low oxygen environment, a humidified hypoxic chamber (Biospherix Redfield, New York) was used to condition BSMC. Variables were set at 3%O2/5%CO2 as in our previous study as well as 1%O2/5%CO2 with the balance N2(gas).11Herz DB Aitken K Bägli DJ Collagen directly stimulates bladder smooth muscle cell growth in vitro: regulation by extracellular regulated mitogen activated protein kinase.J Urol. 2003; 170: 2072-2076Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar Normoxic controls were identical to hypoxic conditions, with the exception of oxygen levels, which were at atmospheric levels of 21%O2. BSMCs were pretreated in serum-free EMEM containing 25 mol/L PD98059 (Calbiochem, San Diego, CA) or 5 to 15 ng/ml rapamycin (Calbiochem, San Diego, CA) for 60 minutes before mechanical strain or hypoxia induction. Cells on collagen gels were treated after attachment (3 hours after plating cells) to denatured or native collagen gels to prevent interference with cell attachment. In all BSMC experiments, serum-starved cells were incubated in 3H-thymidine (GE Healthcare, Piscataway, NJ) at 2μCi/ml before conditioning. At the conclusion of each experiment, radiolabeled counts were fixed in ice-cold methanol, precipitated with ice-cold 5% trichloroacetic acid, solubilized in 0.4 mol/L NaOH + 0.5% SDS and counted as previously described.9Sabha N Aitken K Lorenzo AJ Szybowska M Jairath A Bägli DJ Matrix metalloproteinase-7 and epidermal growth factor receptor mediate hypoxia-induced extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activation and subsequent proliferation in bladder smooth muscle cells.In Vitro Cell Dev Biol Animal. 2006; 42: 124-133Crossref PubMed Scopus (14) Google Scholar, 10Aitken KJ Block G Lorenzo A Herz D Sabha N Dessouki O Fung F Szybowska M Craig L Bägli DJ Mechanotransduction of extracellular signal-regulated kinases 1 and 2 mitogen-activated protein kinase activity in smooth muscle is dependent on the extracellular matrix and regulated by matrix metalloproteinases.Am J Pathol. 2006; 169: 459-470Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar As previously described,9Sabha N Aitken K Lorenzo AJ Szybowska M Jairath A Bägli DJ Matrix metalloproteinase-7 and epidermal growth factor receptor mediate hypoxia-induced extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activation and subsequent proliferation in bladder smooth muscle cells.In Vitro Cell Dev Biol Animal. 2006; 42: 124-133Crossref PubMed Scopus (14) Google Scholar, 10Aitken KJ Block G Lorenzo A Herz D Sabha N Dessouki O Fung F Szybowska M Craig L Bägli DJ Mechanotransduction of extracellular signal-regulated kinases 1 and 2 mitogen-activated protein kinase activity in smooth muscle is dependent on the extracellular matrix and regulated by matrix metalloproteinases.Am J Pathol. 2006; 169: 459-470Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar Western blotting was performed against whole cell lysates or tissue lysates isolated by crushing tissue under N2(liq). Antibodies for blotting comprised phospho-specific antibodies for threonine389-ribosomal S6K, serine235/236-S6, threonine197/202-MNK1, serine65-4EBP, -EIF4<, tyrosine705-STAT3 (all at 1:1000; Cell Signaling Danvers, MA), and SMA (Abcam Cambridge, MA, 1:500). Bands were normalized to total actin (Sigma St. Louis, MO), total p70 S6K or pan-ERK1/2 (1:500; Cell Signaling). Densitometric analysis was performed with Image J as described.9Sabha N Aitken K Lorenzo AJ Szybowska M Jairath A Bägli DJ Matrix metalloproteinase-7 and epidermal growth factor receptor mediate hypoxia-induced extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activation and subsequent proliferation in bladder smooth muscle cells.In Vitro Cell Dev Biol Animal. 2006; 42: 124-133Crossref PubMed Scopus (14) Google Scholar, 10Aitken KJ Block G Lorenzo A Herz D Sabha N Dessouki O Fung F Szybowska M Craig L Bägli DJ Mechanotransduction of extracellular signal-regulated kinases 1 and 2 mitogen-activated protein kinase activity in smooth muscle is dependent on the extracellular matrix and regulated by matrix metalloproteinases.Am J Pathol. 2006; 169: 459-470Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar As described in Herz et al (2003),11Herz DB Aitken K Bägli DJ Collagen directly stimulates bladder smooth muscle cell growth in vitro: regulation by extracellular regulated mitogen activated protein kinase.J Urol. 2003; 170: 2072-2076Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar cells were fixed in ice-cold methanol or 4% paraformaldehyde, and permeabilized with 0.2% Triton-X 100. BSMCs were blocked with 5% goat serum and stained with anti-SMA-Cy3 (1:200; Sigma). Nuclei were counterstained with Hoechst and cells mounted in Dako fluorescent mounting medium. Cells transfected with rat HA-S6K1 plasmids (From Addgene Cambridge, MA)18Fingar DC Richardson CJ Tee AR Cheatham L Tsou C Blenis J mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E.Mol Cell Biol. 2004; 24: 200-216Crossref PubMed Scopus (709) Google Scholar were double-stained for the hemaglutinin (HA) tag using a mouse monoclonal anti-HA antibody (Covance, Princeton, NJ), and a rabbit polyclonal anti-SMA antibody (Abcam) and secondary goat anti-rabbit-Cy3 and goat anti-mouse-Cy2, respectively (both 1:200; Jackson Immunolabs). Nuclei were counterstained with Hoechst. Bladders from Sprague-Dawley 100 to 120 g female rats were mechanically strained by distension during ex vivo whole organ culture for the indicated times (0 to 120 minutes). To perform ex vivo distension, bladders were first catheterized in vivo under anesthesia. Ureters were ligated, and the urethra sutured around the catheter tightly five times. Bladders were distended to 40 cm of hydrostatic pressure by manometry, as described in Capolicchio et al.22Capolicchio G Aitken KJ Gu JX Reddy P Bägli DJ Extracellular matrix gene responses in a novel ex vivo model of bladder stretch injury.J Urol. 2001; 165: 2235-2240Abstract Full Text Full Text PDF PubMed Google Scholar, 19Halachmi H Sarel S Aitken A Karen K Szybowska S Marta M Sabha S Nesrin N Dessouki D Shariff S Lorenzo L Armando A Tse T Derrick D Bagli B Darius D Role of signal transducer and activator of transcription 3 (STAT3) in stretch injury to bladder smooth muscle cells.Cell Tissue Res. 2006; 326: 149-158Crossref PubMed Scopus (23) Google Scholar We found that 40 cm hydrostatic pressure induces strain injury in the bladder, sufficient to alter ECM gene expression,22Capolicchio G Aitken KJ Gu JX Reddy P Bägli DJ Extracellular matrix gene responses in a novel ex vivo model of bladde
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