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

Abstract

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.( 1,2) 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.

Figure 1

Integrated Pathway Analysis of focus genes from studies of strain and hypoxia stimulated bladder SMC. Genes identified through our own work or by microarray analysis were entered into the Ingenuity Pathway Analysis program (Ingenuity Systems, Inc.). The pathway (A) was generated by analyzing the focus genes or proteins (nodes) identified through the literature on strain (red) and/or hypoxia (blue) mediated injury in BSMC and ex vivo bladders using integrated pathway analysis on IPA. The two most significant networks associated with these genes as proposed by IPA are shown (B and C) with the focus genes from (A) highlighted as before. Table 3 shows these pathways, list of genes, network z-scores and functions. Due to the nature of the database, some nodes are duplicated due to changes in the nomenclature of the gene, eg, MAP2K1/2 and Mek, and ERK and MAPK1/3. Nodes or genes highlighted in green were identified as through the IPA curated database as genes relevant to these networks. The networks (B and C) were queried for druggable targets using information on each target in the IPA database. Targets with clinically approved pharmacotherapeutic agents affecting them were circled in green (new to bladder) or red (previously examined in bladder). In (C), IPA uncovered a target previously unstudied in bladder smooth muscle:S6K, a target of rapamycin through its association with mTOR. Other targets include those studied in the context of the bladder, such as the cyclooxygenase-2 pathway (PTGS2 and CYCS), MMPs, the EGF receptor pathway (HBEGF), NMDA receptor, Insulin, and NFAT/Calcineurin, which are circled in red. Further targets may become apparent as the database of IPA increases. The edges (relationships) between each of the nodes (genes) can be queried in an interactive html format of these images (available in online Supplemental Figure S1 at http://ajp.amjpathol.org.). IgE, TCR, FSH, CD3, and other nodes possibly unexpressed in BSMC were omitted from the figures of the networks (full network list available in Table 3).

Figure 2

Distension of intact ex vivo bladder activates effectors of the mTOR Pathway. A: S6 kinase and ERK1/2 phosphorylation was detected in distended ex vivo bladders by immunofluorescence on cryosections using monoclonal anti-phospho-S6 kinase and -phospho-ERK1/2 antibodies (Cell Signaling) and secondary anti-mouse-Cy3 (red). Phospho-S6 activation was also examined with polyclonal anti-phospho-S6 and anti-rabbit-Cy2 (green) with Hoechst (blue) nuclear counterstaining. Localization of mTOR pathway and ERK1/2 activation was examined over a time course from 0 to 120 minutes, revealing a rise in S6K at 30 minutes and gradual increases in both ERK and S6 activation over the longer term. Original magnification, ×200. Scale bar = 80 um. Representative photos of n = 3 bladders. Yellow arrow = phospho-ERK positive vessels in the mucosa. White arrow = phospho-ERK positive suburothelial compartment. Immunofluorescent intensities of the detrusor muscle from N = 3 bladders were analyzed on ImageJ, in individual channels. B: ERK increased in phosphorylation early (5 minutes, *P < 0.05 by two-tailed t-test). Both activated S6 (long dashed line) and ERK (straight line) appeared to increase past 60 and 120 minutes of distension (**P < 0.05, by t-test; ***P < 0.05, by two-tailed t-test, respectively). Phosphorylation of S6K (short dashed line) was increased at 30 minutes of distension, ****P < 0.05 by two-tailed t-test.

Figure 3

Rapamycin inhibits proliferation in response to mitogenic stimuli in BSMC. A: Hypoxia (3% O₂) for 18 hours induced proliferation of BSMC as compared with normoxia (21%O₂) by ³H-thymidine incorporation. Hypoxia was induced using the Pro-ox controller in a humidified hypoxia chamber (Biospherix). Rapamycin (5 ng/ml) did not inhibit control levels of BSMC proliferation, but hypoxia-induced BSMC proliferation was significantly inhibited by rapamycin (*P < 0.05, by 2-factor analysis of variance, **P < 0.04). Each group represents means (±SD) from n = 6. B: Proliferation of BSMC in response to strain for 16 hours is inhibited by rapamycin. Quiescent BSMC at 60 to 70% confluency were incubated ± rapamycin and strained (or not). Elongation was performed with a static pattern, slowly ramping strain up from 2% for 1 hour, 4% 1 hour to 5% for 14 hours increasing slowly to a 5% elongation. Under strain conditions, rapamycin showed a significant inhibitory affect on BSMC proliferation (* vs. others, P < 0.0001, by 2-factor analysis of variance). There was no significant difference between samples without strain ± rapamycin (**P = 0.4303). Each group represents a mean (±SD) of n = 6. C: Denatured matrix induced proliferation is inhibited by rapamycin in BSMCs. Cell counting revealed augmented proliferation on denatured collagen type I matrices, as reported previously. This increase in proliferation was inhibited by rapamycin (* vs. others, P < 0.04, by one-way analysis of variance, n = 3 gels with 10 fields counted each). D: Hypoxia and mechanical strain synergize to increase proliferation of SMCs. BSMC serum-starved and pretreated with rapamycin, were stimulated with nothing, 4% O₂, 5% static mechanical strain for 1 hour, or both 5% strain and 4%O₂ hypoxia, n = 3. The strain and hypoxia parameters each induced lower increases of proliferation individually than seen with higher degrees of these stimuli (*P < 0.01). However, in combination, the milder hypoxia and milder strain induced a significant increase in proliferation compared with the individual stimuli (*P < 0.01, **P < 0.05, ***P < 0.001). All stimulated groups (strain, hypoxia and hypoxia plus strain) were inhibited by rapamycin treatment (P < 0.01, in all cases). Analysis by two-tailed t-test.

Figure 4

Mechanical strain and hypoxia activates S6K downstream of mTOR. BSMCs plated on collagen type I Bioflex plates were serum starved for 48 hours before stimulating with static strain and/or hypoxia. S6K phosphorylation synergistically increased in response to 5% strain in combination with hypoxia. Hypoxia was performed using mixed gas to replace the oxygen in the atmosphere, lowering O₂ to 3%. Combinations of strain with hypoxia were performed using a unique chamber (Biospherix) designed for the use of the Flexcell baseplates in combination with hypoxia. Protein harvested after indicated time points was analyzed by Western blotting for phospho-S6K, total S6K, total actin. Representative autoradiographs from n = 3 blots shown. Densitometry on n = 3 blots was statistically analyzed by two-factor analysis of variance *P < 0.02, **P = 0.0007; ***P < 0.002, n = 3.

Figure 5

Mechanical strain activates many downstream effectors of mTOR. A−C: Downstream effectors were examined in response to strain alone. Cells were statically strained on the Flexcell 4000 system with 5% elongation for A, C: 20 minutes; B: 10 minutes. Whole cell lysates harvested after indicated time points were analyzed by Western blotting for phospho-S6, - MNK1, -ERK1/2, -STAT3, -EIF4E, total actin, and pan-ERK1/2. Representative autoradiographs from n = 3 blots shown. Densitometry on n = 3 blots was statistically analyzed by: (A), phospho-S6: t-test, *P < 0.10; phospho-MNK1: t-test, * vs. **P < 0.05, n = 3; (B), phospho- ERK: t-test, *P < 0.02, n = 3; (C), phospho-STAT3: t-test, *P < 0.04, **P < 0.03, n = 3; phospho- EIF4E: t-test, * vs. **P < 0.04, n = 3.

Figure 6

Rapamycin reverses strain-, hypoxia- and denatured matrix-induced loss of differentiation phenotype. A: Loss of SMA mRNA during strain is significantly restored by rapamycin (*P < 0.05, by t-test). SMA mRNA was assessed by real-time PCR via the deltaC(t) method: the change in expression is compared with housekeeping genes (Rpl32 and Gapdh), then to control unstrained levels. B: BSMCs were plated on collagen type I Flexcell plates and statically strained for 18 hours. Cells were fixed in 4% paraformaldehyde, and stained for SMA using anti-SMA-Cy3 (Sigma) and Hoechst after permeabilization with 0.2% TritonX-100. Loss of SMA expression during strain is restored by rapamycin treatment. C: SMA staining and morphology is altered in denatured matrix-stimulated and strained BSMCs. Serum-starved BSMCs were cultured on denatured collagen versus native collagen gels for 24 hours, with and without rapamycin treatment BSMCs on DNC plates had significantly lower SMA expression (*P < 0.005, by t-test, using a total of n = 4), which was partially recovered by rapamycin treatment (**P = 0.03, by t-test, using a total of n = 3). D:Sma expression is increased in BSMC treated with rapamycin ± hypoxia (1% O₂). Sma expression was assessed by real-time PCR using the deltaC(t) method, where the change in expression is compared with housekeeping genes (Rpl32 and Gapdh), and compared with control normoxic levels.

Figure 7

S6K1 overexpression in BSMCs is associated with decreased SMA expression. BSMCs were plated at 50% confluency in six-well plates, and transfected with rat hemaglutinin(HA)-tagged S6K1 constructs (Addgene) using LTX with plus reagent in OptiMem. After 4 hours, media was replaced with growth media and cells incubated for 2 days. HA-S6K1 was detected using mouse anti-HA antibody (Covance) and anti-mouse-Cy2. SMA was detected using rabbit anti-SMA (Abcam) and anti-rabbit-Cy3. HA expression (a tag for transgene expression) particularly in cells transfected with the constitutively active mutants (D3E E389) was associated with a down-regulation of SMA staining. The cells with the highest HA- tag immunoreactivity (S6K expression) had significantly lower SMA expression compared with low or nontransfected cells (*P < 0.002, **P < 0.007, by two-tailed t-test; data are presented as means ± SEM).

Figure 8

Rapamycin reduces Mmp7 expression induced by mitogenic stimuli. Real-time PCR was performed on cDNA from BSMC, plated on Bioflex plates that were serum-starved and stimulated by 5% equibiaxial strain ± 1% O₂ hypoxia, for 18 hours. Strain plus hypoxia induced a significant upregulation of Mmp7 as compared with unstimulated cells (*P < 0.0008), rapamycin treated or hypoxia stimulated cells (**P < 0.003) and strained cells (^##P = 0.05). Hypoxia (1% O₂) alone did not increase Mmp7 mRNA levels, though in previous work 3% O₂ increased Mmp7 transcription. A trend toward increased Mmp7 was seen in the strain alone group (***P = 0.06), which was decreased significantly in the presence of rapamycin (^#P < 0.04). Data are presented as means ± SEM (n = 3) and P values calculated using 3-factor analysis of variance.

Figure 9

In vivo partial bladder outlet obstruction (PBO) is associated with decreased SMA and increased Mmp7 expression. Outlet obstructions were performed by ligation of the urethra and tube, and removal of the tube, to cause a permanent partial obstruction of the urethra (n = 4). Sham obstructed bladders (n = 3) were not ligated, but all other manipulations were performed. Harvested tissue was crushed under liquid nitrogen for RNA isolation, cDNA synthesized using Superscript III (Invitrogen) and real-time PCR performed using SyBr green. The delta C(t) method of quantitation of real-time PCR results of Mmp7, Sma, and Gapdh (housekeeping gene) revealed that: (A) Mmp7 mRNA was up-regulated during PBO (*P < 0.002, by t-test), and (B) Sma mRNA was down-regulated during PBO (*P = 0.05, by t-test). The pattern of up-regulated Mmp7 and down-regulated Sma recapitulates the in vitro data observed with strain and hypoxia. C: SMA protein expression is down-regulated during PBO, by Western blotting (Sigma monoclonal against SMA), *P < 0.05 by t-test. D: mTOR pathway is activated in vivo during PBO. Activation of S6K and 4EBP was assessed by Western blotting using phospho-specific antibodies to probe tissue lysates harvested by crushing under N2 (liq). Increased phosphorylation of S6K (P = 0.01) and decreased phosphorylation of 4EBP (P = 0.05) were indicative of activation of the two main arms of translation control during PBO (by two-tailed t-test, n = 3).

Figure 10

Model of pathway induction after pathophysiologic stimulation of BSMC by three coordinate stimuli: strain, hypoxia, and damaged matrix. Bladder obstruction leads to strain injury of the bladder smooth muscle. Strain injury is associated with microvascular compression and consequent hypoxia as well as matrix metalloprotease activation and consequent alteration of the ECM. These three coordinate stimuli, hypoxia, strain (directly) and damaged matrix can lead to increased signaling through mTOR, inhibited by rapamycin, as well as parallel pathways ERK, JAK2/STAT3 and p38. EIF4E is strain-activated, but not in an mTOR dependent manner, suggesting that in BSMC strain activates EIF4E through other pathways (EGFR-dependent, p38-dependent), which prevent inhibition by rapamycin under strain 4EBP upstream of EIF E and downstream of mTOR is inhibited by in vivo obstruction in contrast to strain in vitro. (see Discussion). STAT3 phosphorylation is also induced by strain, but is also basally induced by rapamycin. Phosphorylation of S6K on the other hand is augmented by strain, hypoxia, and, as with S6, is inhibited by rapamycin. S6K appears to initiate the de-differentiating response. However, STAT3, ERK and S6K inhibition all prevent proliferation in response to obstruction-related stimuli in BSMC, suggesting a common intermediary in all three pathways.