Calpain-2 Activates Akt via TGFβ1-mTORC2 Pathway. in Pulmonary Artery Smooth Muscle Cells

Transcription

1 Articles in PresS. Am J Physiol Cell Physiol (April 20, 2016). doi: /ajpcell Calpain-2 Activates Akt via TGFβ1-mTORC2 Pathway in Pulmonary Artery Smooth Muscle Cells Prasanna Abeyrathna 1, Laszlo Kovacs 1, Weihong Han 1, and Yunchao Su 1,2,3, Department of Pharmacology & Toxicology, 2 Department of Medicine, 3 Vascular Biology Center, Medical College of Georgia, Augusta University, Augusta, GA 30912; 4 Service, Charlie Norwood Veterans Affairs Medical Center, Augusta, Georgia Research Running Title: Activation of Akt by Calpain Corresponding author: Yunchao Su, MD, Ph.D., Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, th Street, Augusta, GA Tel: (706) Fax: (706) ysu@augusta.edu Copyright 2016 by the American Physiological Society.

2 Abstract Calpain is a family of calcium-dependent nonlysosomal neutral cysteine endopeptidases. Akt is a serine/threonine kinase that belongs to AGC kinases and plays important roles in cell survival, growth, proliferation, angiogenesis, and cell metabolism. Both calpain and Akt are the downstream signaling molecules of platelet-derived growth factor (PDGF) and mediate PDGFinduced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. We found that inhibitions of calpain-2 by using calpain inhibitor MDL28170 and calpain-2 sirna attenuated Akt phosphorylations at serine-473 (S473) and threonine-308 (T308) as well as collagen synthesis and cell proliferation of PASMCs induced by PDGF. Overexpression of calpain-2 in PASMCs induced dramatic increases in Akt phosphorylations at S473 and T308. Moreover, knockout of calpain attenuated Akt phosphorylations at S473 and T308 in smooth muscle of pulmonary arterioles of mice with chronic hypoxic pulmonary hypertension. The cell-permeable specific TGFβ receptor inhibitor SB attenuated Akt phosphorylations at both S473 and T308 induced by PDGF and by overexpressed calpain-2 in PASMCs. Furthermore, SB and knocking down ALK5 significantly reduced PDGF-induced collagen synthesis and cell proliferation of PASMCs. Nevertheless, neutralizing extracellular TGFβ1 using a cell-impermeable TGFβ1 neutralizing antibody did not affect PDGF-induced Akt phosphorylations at S473 and T308. Further, inhibition of mtorc2 by knocking down its component protein Rictor prevented Akt phosphorylations at S473 and T308 induced by PDGF and by overexpressed calpain-2. These data provide first evidence supporting that calpain-2 up-regulates PDGF-induced Akt phosphorylation in pulmonary vascular remodeling via an intracrine TGFβ1/mTORC2 mechanism. 2

3 Key words: PDGF, pulmonary hypertension, vascular smooth muscle cells, Rictor Introduction Pulmonary vascular remodeling is a key feature of pulmonary arterial hypertension (PAH) associated with accumulation of extracellular matrix, including collagen, and vascular smooth muscle cell proliferation and hypertrophy. These processes contribute to medial hypertrophy and muscularization leading to obliteration of precapillary pulmonary arteries and sustained elevation of pulmonary arterial pressure (31; 32; 39). Several growth factors including PDGF and TGF-β1 participate in the process of pulmonary vascular remodeling in PAH patients and animal models (3; 11; 18; 27; 32). For example, PDGF and its receptor is up-regulated in pulmonary arteries of patients with PAH (34; 35) and rodents exposed to chronic hypoxia and monocrotaline (MCT) (3; 28; 41). PDGF receptor antagonist prevents or reverses increased right ventricular pressure and pulmonary vascular changes induced by hypoxia, MCT, and hypoxia/su5416 (8; 34). TGFβ1/Smad pathway is a classical pathway regulating collagen synthesis. It has been reported that TGF-β1/Smad pathway is activated in PAH animal models (3; 27; 28) and patients with PAH (38). Inhibition of TGF-beta signaling attenuates pulmonary vascular remodeling and elevated right ventricular pressure in animal models (6; 27; 40). Despite of these overwhelming data, approaches for intervention of these growth factors are limited, because the down-stream signaling pathways of these growth factor receptors have not been clarified. Calpain is a family of calcium-dependent nonlysosomal neutral cysteine endopeptidases (14). Calpain-1 and calpain-2 are two major typical calpains, which consist of a distinct large catalytic subunit (about 80 kda) and a common small subunit (about 30 kda, calpain-4) that helps regulate activity. Calpastatin functions as the major specific endogenous inhibitor for calpain-1 and calpain-2 (14). The mechanism for calpain activation involves calcium, phospholipid 3

4 binding, autolysis, release of calpain from its inhibitor calpastatin, binding of activator proteins and phosphorylation (12; 13). Binding of phospholipids may decrease the Ca 2+ requirement for calpain-2 activation (4). We have reported that calpain is a downstream signal molecule for PDGF and mediates PDGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs) (28). Inhibition of calpain by conditional knockout of calpain-4 and by using calpain inhibitor prevents pulmonary vascular remodeling in PAH animal models (28). Akt is a serine/threonine kinase that belongs to AGC kinases. Akt plays important roles in cell survival, growth, proliferation, angiogenesis, and cell metabolism (1). It works as a downstream signaling molecule for several growth factors including PDGF (10; 26; 30). Phosphorylation of Akt at serine-473 (S473) or threonine-308 (T308) causes activation of Akt (2). T308 residue is phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) due to its association with PtdIns (3,4,5)P3, while mammalian target of rapamycin 2 (mtor2) is responsible for the phosphorylation of S473 residue (15; 29). Akt plays an essential role in pulmonary vascular remodeling and the development of pulmonary hypertension (36). mtorc2 and P-Akt-S473 are increased in PASMCs of human lung with PAH and in PASMCs exposed to hypoxia and PDGF (15; 23). Knockout of Akt1 and mtor attenuate pulmonary vascular remodeling of hypoxic PAH (36). Inhibition of mtoc using rapamycin prevents PAH in MCT rat model (20). Moreover, activations of Akt and mtor are required for hypoxia-induced cell proliferation of vascular endothelial cells and adventitial fibroblasts of pulmonary arteries (9; 26). Nevertheless, the detail mechanism for the phosphorylation and activation of Akt in pulmonary vascular remodeling of PAH remains unclear. 4

5 Because both calpain and Akt work at the downstream signaling of PDGF, we hypothesize that there might be a connection between calpain activation and Akt in the collagen synthesis and cell proliferation of PASMCs caused by PDGF. In the present study, we demonstrate that inhibition of calpain-2 causes a reduction of PDGF-induced Akt phosphorylation and that overexpression of calpain-2 induces an increase in Akt phosphorylation. We found, for the first time, that calpain-2 up-regulates Akt phosphorylation via an intracrine TGFβ1/mTORC2 mechanism Materials and Methods Materials Human PASMCs and culture medium were obtained from Lonza (Walkersville, MD). MDL28170 was obtained from Calbiochem (San Diego, CA). SB was from Tocris (Ellisville, MO). Anti-GAPDH, anti-p-akt-s473, anti-p-akt-t308, anti-total Akt, anti-gapdh antibodies, Akt sirna, LY294002, and BrdU cell proliferation kit were purchased from Cell Signaling Technology (Beverly, MA). Antibody against collagen-i was from Novus Biologicals (Littleton, CO). Anti-calpain-1 and -2 antibodies were purchased from Triple Point Biologics (Forest Grove, OR). Anti-Rictor antibody is from Bethyl Laboratories (Montgomery, TX). Anti- ALK5 antibody and ALK5 sirna is from Santa Cruz Biotechnology (Dallas, TX). PDGF-BB and all other regents were obtained from Sigma-Aldrich (St. Louis, MO). Cell culture Human PASMCs were cultured according to the manufacturer s instructions. Prior to all experimentation, the third- to seventh-passage cells were equilibrated in growth factor free medium for 24 h. 5

6 SDS-PAGE and Western blot analysis μg of proteins were resolved in 7.5% or 4-15 % Criterion SDS-polyacrylamide gel electrophoresis (Bio-Rad). Proteins were then transferred to nitrocellulose membrane using the Criterion transfer system. P-Akt-S473, P-Akt-T308, total Akt, collagen-i, ALK5, and Rictor were detected by immunoblotting the membrane using primary antibodies against calpain-1 (1:5000), calpain-2 (1:5000), collagen-i (1:1000), P-Akt-S473 (1:2000), P-Akt-T308 (1:2000), total Akt (1:1000), ALK5 (1:1000), Rictor (1:1000) and GAPDH (1:1000) overnight in TBST with 1% BSA. Bound antibodies were detected using a goat anti-rabbit (or mouse) IgG-AP conjugate (Bio-Rad) at 1:5000 dilution using TBST with 1% BSA. Proteins were detected using an Immun-Star AP chemiluminescence kit (Bio-Rad). Densitometric analysis of the protein bands were performed using Quantity One 1-D Analysis Software. RNA interference in PASMCs The expressions of calpain-1, calpain-2, Akt, ALK5, and Rictor were silenced using sirna technology. Calpain-1 sirna (target sequence: 5 -AAGCTAGTGTTCGTGCACTCT-3 ), calpain-2 sirna (target sequence: 5 -CTGGAACACTATAGACCCAGA-3 ) and the nontargeting sirna (target sequence: 5 - AACGTACGCGGAATACTTCGA-3 ) were synthesized 129 by Qiagen. The Akt sirna (6211) is from Cell Signaling Technology. ALK5 sirna (SC40222) is from Santa Cruz Biotechnology. Rictor sirna is a mix of two Rictor sirnas ( and ) from Qiagen. The sequences of sirna were not disclosed by the companies. The sirnas were transfected into PASMCs with siport amine transfection reagent according to the manufacturer s instruction. 3 days after transfection, the medium was changed to serum-free medium for 24 h followed by the treatments of PASMCs. Overexpression of calpain-2 in PASMCs 6

7 Total RNA was isolated from PASMCs with RNeasy RNA Isolation kit (Qiagen, Valencia, CA), and the mrna was reverse-transcribed with High-Capacity cdna Reverse Transcription Kit (Life Technologies, Grand Island, NY). The cdna was amplified by PCR using Expand High Fidelity PCR System (Roche Applied Science (Indianapolis, IN) as well as calpain-2- forward 5 -AAAAAAAGCTTGGATGGCGGGCATCGCGGC-3 and calpain-2-reverse AAAAATCTAGAAAGTACTGAGAAACAGAGCCAAGAGATAAGGTCG-3 primers (Integrated DNA Technologies, Coralville, IA) containing HindIII and XbaI restrictions sites, respectively. Primers were designed according to the homo sapiens calpain-2 sequence (NCBI 144 database accession number: NM_ ). Then the cdna was cloned into a pcdna TM 3.1/V5-His (Life Technologies, Grand Island, NY) mammalian expression vectors to create the pcdna3.1/v5-his-human calpain-2 wild type plasmid. The vector construct was confirmed by DNA sequencing. PASMCs were transfected with pcdna3.1/v5-his-human calpain-2 wild type construct using X-tremeGENE HP transfection reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturer's protocol. 48 h after the transfection, the medium was changed to serum-free medium for 24 h followed by the treatments of cells. Calpain activity Assay Calpain activity in PASMCs was measured with a SpectraMax M2e microplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA) as described previously (28). Briefly, after the treatments the cells were washed with PBS, and the fluorogenic Suc-Leu-Leu-Val-Tyr- AMC peptide substrate was added to a final concentration of 80 μm in the presence and absence of MDL Fluorescence was immediately recorded at 2-min intervals for 20 min. Calpain activity was calculated as fluorescence units in the absence of MDL28170 subtracted by those in the presence of MDL

8 Cell proliferation assay Cells were cultured in 96 well plates for 24 h and serum-starved for 12 h. A colorimetric BrdU ELISA kit was used. After the treatments, BrdU incorporation was detected by using anti- BrdU peroxidase conjugate antibody according to the manufacturer s instructions. Absorbance at 450 nm was measured by using a Spectra-Max M2e microplate reader (Molecular Devices Corporation, Sunnyvale, CA). Determination of Akt phosphorylation in pulmonary arteries from calpain knockout mice and control mice with chronic hypoxic PAH To investigate the role of calpain in Akt phosphorylation in in pulmonary vascular remodeling in vivo, calpain activity was inhibited in a mouse line of inducible global knock-out of calpain-4 (ER-Cre +/- Capn4 flox/flox ) as described previously (28). The animal experiments were performed in accordance with the guiding principles of the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (IACUC) of the Augusta University. Because calpain-4 is required for the activity of both calpain-1 and -2, deletion of calpain-4 prevents activation of calpain-1 and -2. Deletion of calpain-4 was induced by administration of tamoxifen (20 mg/kg/day, i.p.) for 5 days. Control mice were littermate Capn4 flox/flox or Capn4 flox/+ mice treated with the same tamoxifen regimen. Mice between 12 and 16 weeks old were used for the hypoxic PAH model as described previously (28). To determine the extent of phosphorylated Akt in pulmonary arterioles, double immuno-staining of P-Akt/αactin was performed on lung tissue slides. The slides were incubated first with a rabbit polyclonal antibody against P-Akt-S473, P-Akt-T308 or total Akt and mouse monoclonal antibody against α-actin overnight and then with goat anti-rabbit IgG Alexa Fuor 488 and goat anti-mouse IgG Alexa Fuor 594. The slides were sealed with mounting solution containing anti- 8

9 fade reagent and were examined using a Zeiss LSM 510 laser scanning confocal microscope. The fluorescence intensities in the vascular smooth muscle layer of pulmonary arterioles were measured using ImageJ. Statistical analysis Within each experiment, cells were matched for number of passages to avoid differences related to tissue culture variables. Results are shown as means ± SEM for n experiments. One way ANOVA and t test analysis (2-tailed) were used to determine the significance of differences between the means of experimental and control groups. A value of P < 0.05 was considered significant Results Inhibition of Akt attenuates PDGF-induced collagen synthesis and proliferation of PASMCs Incubation of PASMCs with PDGF-BB (10 ng/ml) for h induces a rapid increase in Akt activation and phosphorylations at S473 and T308 (Figure 1A). The increases in P-Akt-S473 and P-Akt-T308 were at the highest levels at 0.5 and 1 h incubation and last for at least 24 h. To investigate the role of Akt activation in PDGF-induced collagen synthesis and cell proliferation of PASMCs, Akt was inhibited by the specific Akt inhibitor triciribine and by using Akt sirna gene silencing. As shown in Figures 1B, 1C, 1D, 1E, 1F, and 1G, incubation of PASMCs with PDGF-BB (10 ng/ml) for 24 h caused increases in collagen-i protein level and cell proliferation. Triciribine (10 µm) and Akt knockdown significantly attenuated PDGF-induced increases in collagen-i protein level and cell proliferation. These data confirm that Akt plays an important role in PDGF-induced collagen synthesis and cell proliferation of PASMCs. 9

10 Inhibition of calpain-2 prevents PDGF-induced collagen synthesis and proliferation of PASMCs We have previously shown that PDGF causes calpain activation and that calpain-2 rather than calpain-1 is responsible for increases in calpain activities induced by PDGF in PASMCs (22). Here we showed that pan calpain inhibitor MDL28170 (20 µm) prevented PDGF-induced collagen synthesis and cell proliferation of PASMCs (Figures 2A, 2B and 2C). To investigate whether calpain-mediated collagen synthesis and cell proliferation is caused by activation calpain-1 or calpain-2, calpain-1 and calpain-2 were specifically knocked down using their sirna. As shown in Figures 2D, 2E and 2F, knockdown of calpain-2, but not calpain-1, prevented PDGF-induced collagen synthesis and cell proliferation of PASMCs. These data indicate that calpain-2 rather than calpain-1 is responsible for increases in collagen synthesis and cell proliferation induced by PDGF. Inhibition of calpain attenuates Akt phosphorylation in PASMCs As shown in Figures 3A and 3B, incubation of PASMCs with calpain inhibitor MDL28170 prevented PDGF-induced increases in the levels of both P-Akt-S473 and P-Akt-T308. The reduction of PDGF-induced increase in P-Akt-S473 was much greater than those in P-Akt-T308 in MDL28170-treated PASMCs, suggesting that calpain may primarily impact Akt phosphorylation at S473. Consistent with the importance of calpain-2 in PDGF-induced collagen synthesis and cell proliferation, knockdown of calpain-2, but not calpain-1, prevented PDGFinduced increases in the levels of P-Akt-S473 and P-Akt-T308 (Figures 3C and 3D). These results provide first evidence that it is calpain-2 rather than calpain-1 that leads to Akt activation and phosphorylation induced by PDGF in PASMCs. Overexpression of calpain-2 increases the levels of P-Akt-S473 and P-Akt-T308 10

11 To further investigate the role of calpain-2 in Akt phosphorylation, calpain-2 was overexpressed in PASMCs by using a plasmid containing human calpain-2 gene. As shown in Figure 4, the levels of P-Akt-S473 and P-Akt-T308 were much higher in PASMCs transfected with plasmids containing calpain-2 gene than those with empty plasmids. The increase in phosphorylation at S473 is much higher than those at T308. These data further support that calpain-2 signaling leads to Akt phosphorylation in PASMCs. Calpain knockout inhibits Akt phosphorylation in smooth muscle of pulmonary arteries in mice with chronic hypoxic PAH We have reported that knockout of calpain-4 (small unit) attenuates hypoxic pulmonary hypertension and vascular remodeling (22; 28). To investigate the role of calpain in Akt phosphorylation in pulmonary vascular remodeling in vivo, we determined the levels of P-Akt- S473 and P-Akt-T308 in smooth muscle of pulmonary arteries from calpain knockout mice and control mice with chronic hypoxic PAH. As shown in Figure 5, the levels of P-Akt-S473 and P- Akt-T308 in smooth muscle of pulmonary arteries were much higher in control mice exposed to hypoxia than those exposed to normoxia. However, the levels of P-Akt-S473 and P-Akt-T308 in smooth muscle of pulmonary arteries were comparable between calpain knockout mice exposed to normoxia and those exposed to hypoxia. These results indicate that inhibition of calpain by conditional knockout of calpain-4 inhibits Akt phosphorylation in hypoxia-induced pulmonary vascular remodeling in vivo. Inhibition of TGFβ receptor prevents calpain-2-induced increases in the levels of P-Akt- S473 and P-Akt-T308 We have previously reported that calpain cleaves and activates latent TGFβ in vitro and in PASMCs (28). To investigate whether calpain 2 increases the levels of P-Akt-S473 and P-Akt- 11

12 T308 through TGFβ pathway, PASMCs with and without calpain-2 overexpression were incubated with and without specific TGFβ receptor blocker SB (10 µm). As shown in Figure 6, SB attenuated the increases in P-Akt-S473 level and to a less extent in P-Akt- T308 level in calpain 2-overexpressed PASMCs. These results indicate that calpain 2 increases the levels of P-Akt-S473 and P-Akt-T308 through TGFβ pathway. Inhibition of TGFβ receptor, but not neutralizing extracellular TGFβ, reduces PDGFinduced Akt phosphorylation, collagen synthesis and cell proliferation We have previously reported that PDGF does not activate extracellular TGFβ and that calpain activates an intracrine TGFβ pathway induced by PDGF in PASMCs (28). To examine whether this intracrine TGFβ pathway is involved in the calpain-mediated Akt phosphorylation, we first assessed the effect of a cell-impermeable TGFβ neutralizing antibody (2 µg/ml) on Akt phosphorylation. This antibody has been previously shown to be able to block the effect of extracellular TGFβ1 on PASMCs (28). We found that neutralizing extracellular TGFβ did not affect PDGF-induced increases in P-Akt-S473 and P-Akt-T308 (Figure 7). Nevertheless, the cell-permeable SB (10 µm) significantly reduced PDGF-induced increases in P-Akt- S473, P-Akt-T308, collagen-i and cell proliferation of PASMCs (Figure 8). Moreover, knock down of ALK5 prevented PDGF-induced increases in collagen-i protein level and cell proliferation of PASMCs (Figure 9). These results indicate that the intracrine TGFβ pathway is involved in the calpain-mediated Akt phosphorylation in PASMCs. Knock down of Rictor attenuates PDGF- and calpain-2-induced Akt phosphorylation in PASMCs More recent studies revealed the phosphorylation of Akt at S473 is primarily mediated by mtorc2 in Akt (15; 29). To investigate whether mtorc2 contributes PDGF- and calpain-2-12

13 induced Akt phosphorylation, mtorc2 was inhibited by knocking down Rictor protein in this complex. As shown in Figure 10, knock down of Rictor attenuates PDGF-induced Akt phosphorylation at S473 and to a less extent at T308 in PASMCs. Moreover, knock down of Rictor prevented Akt phosphorylations at S473 and T308 in calpain-2-overexpressed PASMCs (Figure 11). These data suggest that mtorc2 contributes to calpain-2-mediated Akt phosphorylation induced by PDGF. PI3 kinase inhibitor LY does not affect PDGF-induced calpain activation and calpain-2-induced Akt phosphorylation To investigate whether PI3 kinase is involved in PDGF-induced calpain activation and calpain-mediated Akt phosphorylation, PI3 kinase was inhibited by using LY As shown in Figure 12A, the PI3 kinase inhibitor LY did not affect PDGF-induced increase in calpain activity. Similarly, LY did not inhibit calpain-2-induced Akt phosphorylations at S473 and T308 (Figures 12B and 12C). These results suggest that PI3 kinase is not involved in PDGF-induced calpain activation and calpain-mediated Akt phosphorylation Discussion Akt is the downstream signaling molecule of PDGF and mediate PDGF-induced collagen synthesis and proliferation of PASMCs, the most critical process in the medial layer thickening in pulmonary vascular remodeling (15; 30). Knocking out Akt1 attenuates the development and progression of pulmonary vascular remodeling and pulmonary hypertension (36). The activation of Akt depends on the phosphorylation of the most important residues at T308 or S473. T308 residue is phosphorylated by PI3 kinase activated PDK1 due to its association with PtdIns (3,4,5)P3, while mtorc2 is responsible for the phosphorylation of S473 residue (29). PDGF 13

14 induces phosphorylation of both T308 and S473 in PASMCs. Akt phosphorylation is increased in PASMCs of human lung with PAH and in PASMCs exposed to hypoxia and PDGF (15; 23). Moreover, activation of Akt is required for hypoxia-induced cell proliferation of vascular endothelial cells and adventitial fibroblasts of pulmonary arteries (9; 26). In the present study, inhibition of Akt significantly attenuates PDGF-induced increases in collagen-i protein level and cell proliferation, confirming that Akt plays an important role in PDGF-induced collagen synthesis and cell proliferation of PASMCs in pulmonary vascular remodeling (30; 36). We have previously reported that calpain is a downstream signal molecule for PDGF (28). PDGF-induced calpain activation is through ERK-mediated calpain phosphorylation (22). We have shown that calpain mediates PDGF-induced collagen synthesis and proliferation of PASMCs (28). Inhibition of calpain by conditional knockout of calpain and by using calpain inhibitor prevents pulmonary vascular remodeling in PAH animal models (22; 28). Our present work here identified a novel link between calpain and Akt phosphorylation in PASMCs. Inhibition of calpain prevents PDGF-induced Akt phosphorylation at S473 and partially blocks PDGF-induced Akt phosphorylation at T308. By separately knocking down calpain-1 and calpain-2 using sirna technology, we found that it is calpain-2 rather than calpain-1 that leads to phosphorylation and activation of Akt, collagen synthesis and cell proliferation of PASMCs induced by PDGF. This is consistent with our previous finding that calpain-2 rather than calpain- 1 is responsible for increases in calpain activity induced by PDGF (22). Interestingly, Ho et al has also shown that mouse mammary carcinoma AC2M2 cells deficient in calpain-2 have lower levels of Akt phosphorylation at S473 (19). We have shown that overexpression of calpain-2 in PASMCs induces dramatic increases in Akt phosphorylations at S473 and T308. Thus, our work 14

15 provides solid evidence, for the first time, showing that calpain-2 up-regulates Akt phosphorylation. We have reported that calpain activation and calpain phosphorylation at S50 are much higher in smooth muscle of pulmonary arteries in rodents of PAH models and patients with PAH (22; 28). Consistently, in the present study, Akt phosphorylations at S473 and T308 are also increased in smooth muscle of pulmonary arteries in mice with hypoxic PAH. Interestingly, inhibition of calpain activity by using knockout of calpain-4 dramatically reduces Akt phosphorylations at S473 and T308 in smooth muscle of pulmonary arteries in mice with hypoxic PAH. These findings provide further evidence supporting that calpain-2 up-regulates Akt phosphorylation in pulmonary vascular remodeling of PAH. PDGF activates PI3 kinase, leading to the interaction of Akt and PDK1 that causes Akt phosphorylation (1). Inhibition of PI3 kinase using LY attenuates PDGF-induced Akt phosphorylation and activation (7; 26). We found here that inhibition of PI3 kinase using LY did not affect PDGF-induced calpain activation. Similarly, LY did not inhibit calpain-2-induced Akt phosphorylation. Therefore, the calpain-mediated Akt phosphorylation is independent of PI3 kinase. On the other hand, PDGF-induced collagen synthesis and proliferation is through an intracellular activation of TGFβ1 in PASMCs (28). Calpain cleaves and activates intracellular latent TGFβ1and initiates this intracrine TGFβ1 pathway (5; 16; 28). It has been reported that TGFβ activates Akt in many types of cells (21; 25; 37). We examined whether this intracrine TGFβ pathway is involved in the calpain-mediated Akt phosphorylation. We found that neutralizing extracellular TGFβ1 using a cell-impermeable TGFβ1 neutralizing antibody did not affect PDGF-induced Akt phosphorylations at S473 and T308, suggesting that the paracrine or autocrine TGFβ pathways do not play a role in calpain-mediated Akt 15

16 phosphorylation. Nevertheless, the cell-permeable specific TGFβ receptor blocker SB attenuated Akt phosphorylations at both S473 and T308 induced by overexpressed calpain-2 and PDGF in PASMCs. Moreover, SB and knocking down ALK5 significantly reduced PDGF-induced collagen synthesis and cell proliferation of PASMCs. These results indicate that calpain 2 up-regulates Akt phosphorylation via an intracrine TGFβ1 pathway. Hart and Vogt discovered that mimicking phosphorylation at S473 by mutating S473 for aspartate leads to an enhancement of phosphorylation at T308 (17). We noted that the increase in phosphorylation at S473 is much higher than those at T308 in calpain-2-overexpressed cells. Moreover, the reduction of PDGF-induced Akt phosphorylation caused by calpain inhibition is much more remarkable at S473 than those at T308. Similarly, the reduction of PDGF- and calpain-2-induced Akt phosphorylation caused by TGFβ inhibition is much greater at S473 than those at T308. These results suggest that phosphorylation of Akt at S473 might be a primary event. The TGFβ1 signaling may first phosphorylate Akt at S473 that is followed by Akt binding to PDK1 and phosphorylation at T308 ensues. Akt phosphorylation at S473 is mediated by mtorc2 (15; 29). Indeed, mtorc2 is at the down-stream of TGFβ receptor and mediates the fibrotic effects of TGFβ1 (24; 33). mtorc2 is increased in PASMCs of human lung with PAH and in PASMCs exposed to hypoxia and PDGF (15; 23). Activations of mtor and Akt are required for hypoxia-induced cell proliferation of vascular endothelial cells and adventitial fibroblasts of pulmonary arteries (9; 26). Our studies show that inhibition of mtorc2 by knocking down its component protein Rictor prevents PDGF- and calpain-2-induced Akt phosphorylation at S473 and attenuates in a less magnitude Akt phosphorylation at T308. Taken together, our data provide first evidence supporting that calpain-2 up-regulates Akt phosphorylation via mtorc2. 16

17 In summary, we have demonstrated that overexpression of calpain-2 induces an increase in Akt phosphorylation and that inhibition of calpain-2 causes a reduction of Akt phosphorylation in PASMCs induced by PDGF and in hypoxic PAH mice. Inhibition of TGFβ signaling attenuates Akt phosphorylation induced by PDGF and by overexpressed calpain-2 in PASMCs. Neutralizing extracellular TGFβ1 using a cell-impermeable TGFβ1 neutralizing antibody does not affect PDGF-induced Akt phosphorylation. Thus, we have identified a novel pathway of calpain-2 up-regulated Akt phosphorylation via an intracrine TGFβ1/mTORC2 mechanism that is independent of PI3 kinase (Figure 13) Sources of Funding This work was supported, in whole or in part, by NIH grants HL and HL115078, Flight Attendants Medical Research Institute grant _CIA and _CIA, and by the Department of Veterans Affairs Authorship Contributions Participated in research design: Prasanna Abeyrathna and Yunchao Su. Conducted experiments: Prasanna Abeyrathna, Laszlo Kovacs and Weihong Han. Performed data analysis: Prasanna Abeyrathna, Laszlo Kovacs and Yunchao Su Wrote or contributed to the writing of the manuscript: Prasanna Abeyrathna and Yunchao Su

18 References Abeyrathna P and Su Y. The critical role of Akt in cardiovascular function. Vascul Pharmacol 74: 38-48, Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P and Hemmings BA. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 15: , Arcot SS, Lipke DW, Gillespie MN and Olson JW. Alterations of growth factor transcripts in rat lungs during development of monocrotaline-induced pulmonary hypertension. Biochem Pharmacol 46: , Arora AS, de Groen PC, Croall DE, Emori Y and Gores GJ. Hepatocellular carcinoma cells resist necrosis during anoxia by preventing phospholipase-mediated calpain activation. J Cell Physiol 167: , Caver TE, O'Sullivan FX, Gold LI and Gresham HD. Intracellular demonstration of active TGFbeta1 in B cells and plasma cells of autoimmune mice. IgG-bound TGFbeta1 suppresses neutrophil function and host defense against Staphylococcus aureus infection. J Clin Invest 98: ,

19 Chen YF, Feng JA, Li P, Xing D, Zhang Y, Serra R, Ambalavanan N, Majid-Hassan E and Oparil S. Dominant negative mutation of the TGF-beta receptor blocks hypoxiainduced pulmonary vascular remodeling. J Appl Physiol 100: , Choi KH, Kim JE, Song NR, Son JE, Hwang MK, Byun S, Kim JH, Lee KW and Lee HJ. Phosphoinositide 3-kinase is a novel target of piceatannol for inhibiting PDGF-BBinduced proliferation and migration in human aortic smooth muscle cells. Cardiovasc Res 85: , Ciuclan L, Hussey MJ, Burton V, Good R, Duggan N, Beach S, Jones P, Fox R, Clay I, Bonneau O, Konstantinova I, Pearce A, Rowlands DJ, Jarai G, Westwick J, Maclean MR and Thomas M. Imatinib attenuates hypoxia-induced pulmonary arterial hypertension pathology via reduction in 5-hydroxytryptamine through inhibition of tryptophan hydroxylase 1 expression. Am J Respir Crit Care Med 187: 78-89, Gerasimovskaya EV, Tucker DA and Stenmark KR. Activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin is necessary for hypoxia-induced pulmonary artery adventitial fibroblast proliferation. J Appl Physiol (1985 ) 98: , Ghosh CG, Lenin M, Calhaun C, Zhang JH and Abboud HE. PDGF inactivates forkhead family transcription factor by activation of Akt in glomerular mesangial cells. Cell Signal 15: , Gillespie MN, Rippetoe PE, Haven CA, Shiao RT, Orlinska U, Maley BE and Olson JW. Polyamines and epidermal growth factor in monocrotaline-induced pulmonary hypertension. Am Rev Respir Dis 140: ,

20 Glading A, Bodnar RJ, Reynolds IJ, Shiraha H, Satish L, Potter DA, Blair HC and Wells A. Epidermal growth factor activates m-calpain (calpain II), at least in part, by extracellular signal-regulated kinase-mediated phosphorylation. Mol Cell Biol 24: , Glading A, Lauffenburger DA and Wells A. Cutting to the chase: calpain proteases in cell motility. Trends Cell Biol 12: 46-54, Goll DE, Thompson VF, Li H, Wei W and Cong J. The calpain system. Physiol Rev 83: , Goncharov DA, Kudryashova TV, Ziai H, Ihida-Stansbury K, DeLisser H, Krymskaya VP, Tuder RM, Kawut SM and Goncharova EA. Mammalian target of rapamycin complex 2 (mtorc2) coordinates pulmonary artery smooth muscle cell metabolism, proliferation, and survival in pulmonary arterial hypertension. Circulation 129: , Gressner OA, Lahme B, Siluschek M, Rehbein K, Herrmann J, Weiskirchen R and Gressner AM. Activation of TGF-beta within cultured hepatocytes and in liver injury leads to intracrine signaling with expression of connective tissue growth factor. J Cell Mol Med 12: , Hart JR and Vogt PK. Phosphorylation of AKT: a mutational analysis. Oncotarget 2: , Hassoun PM, Mouthon L, Barbera JA, Eddahibi S, Flores SC, Grimminger F, Jones PL, Maitland ML, Michelakis ED, Morrell NW, Newman JH, Rabinovitch M, Schermuly R, Stenmark KR, Voelkel NF, Yuan JX and Humbert M. Inflammation, growth factors, and pulmonary vascular remodeling. J Am Coll Cardiol 54: S10-S19,

21 Ho WC, Pikor L, Gao Y, Elliott BE and Greer PA. Calpain 2 regulates Akt-FoxOp27(Kip1) protein signaling pathway in mammary carcinoma. J Biol Chem 287: , Houssaini A, Abid S, Mouraret N, Wan F, Rideau D, Saker M, Marcos E, Tissot CM, Dubois-Rande JL, Amsellem V and Adnot S. Rapamycin reverses pulmonary artery smooth muscle cell proliferation in pulmonary hypertension. Am J Respir Cell Mol Biol 48: , Kattla JJ, Carew RM, Heljic M, Godson C and Brazil DP. Protein kinase B/Akt activity is involved in renal TGF-beta1-driven epithelial-mesenchymal transition in vitro and in vivo. Am J Physiol Renal Physiol 295: F215-F225, Kovacs L, Han W, Rafikov R, Bagi Z, Offermanns S, Saido TC, Black SM and Su Y. Activation of calpain-2 by mediators in pulmonary vascular remodeling of pulmonary arterial hypertension. Am J Respir Cell Mol Biol 54: , Krymskaya VP, Snow J, Cesarone G, Khavin I, Goncharov DA, Lim PN, Veasey SC, Ihida-Stansbury K, Jones PL and Goncharova EA. mtor is required for pulmonary arterial vascular smooth muscle cell proliferation under chronic hypoxia. FASEB J 25: , Lamouille S, Connolly E, Smyth JW, Akhurst RJ and Derynck R. TGF-beta-induced activation of mtor complex 2 drives epithelial-mesenchymal transition and cell invasion. J Cell Sci 125: , Li C, Wang Q and Wang JF. Transforming growth factor-beta (TGF-beta) induces the expression of chondrogenesis-related genes through TGF-beta receptor II (TGFRII)-AKT- 21

22 mtor signaling in primary cultured mouse precartilaginous stem cells. Biochem Biophys Res Commun 450: , Li L, Xu M, Li X, Lv C, Zhang X, Yu H, Zhang M, Fu Y, Meng H and Zhou J. Platelet-derived growth factor-b (PDGF-B) induced by hypoxia promotes the survival of pulmonary arterial endothelial cells through the PI3K/Akt/Stat3 pathway. Cell Physiol Biochem 35: , Long L, Crosby A, Yang X, Southwood M, Upton PD, Kim DK and Morrell NW. Altered bone morphogenetic protein and transforming growth factor-beta signaling in rat models of pulmonary hypertension: potential for activin receptor-like kinase-5 inhibition in prevention and progression of disease. Circulation 119: , Ma W, Han W, Greer PA, Tuder RM, Toque HA, Wang KKW, Caldwell RW and Su Y. Calpain mediates pulmonary vascular remodeling in rodent models of pulmonary hypertension and its inhibition attenuates pathologic features of disease. J Clin Invest 121: , Moschella PC, McKillop J, Pleasant DL, Harston RK, Balasubramanian S and Kuppuswamy D. mtor complex 2 mediates Akt phosphorylation that requires PKCepsilon in adult cardiac muscle cells. Cell Signal 25: , Ogawa A, Firth AL, Smith KA, Maliakal MV and Yuan JX. PDGF enhances storeoperated Ca2+ entry by upregulating STIM1/Orai1 via activation of Akt/mTOR in human pulmonary arterial smooth muscle cells. Am J Physiol Cell Physiol 302: C405-C411, Rabinovitch M. Pathobiology of pulmonary hypertension. Annu Rev Pathol 2: ,

23 Rabinovitch M. Molecular pathogenesis of pulmonary arterial hypertension. J Clin Invest 118: , Rahimi RA, Andrianifahanana M, Wilkes MC, Edens M, Kottom TJ, Blenis J and Leof EB. Distinct roles for mammalian target of rapamycin complexes in the fibroblast response to transforming growth factor-beta. Cancer Res 69: 84-93, Schermuly RT, Dony E, Ghofrani HA, Pullamsetti S, Savai R, Roth M, Sydykov A, Lai YJ, Weissmann N, Seeger W and Grimminger F. Reversal of experimental pulmonary hypertension by PDGF inhibition. J Clin Invest 115: , Selimovic N, Bergh CH, Andersson B, Sakiniene E, Carlsten H and Rundqvist B. Growth factors and interleukin-6 across the lung circulation in pulmonary hypertension. Eur Respir J 34: , Tang H, Chen J, Fraidenburg DR, Song S, Sysol JR, Drennan AR, Offermanns S, Ye RD, Bonini MG, Minshall RD, Garcia JG, Machado RF, Makino A and Yuan JX. Deficiency of Akt1, but not Akt2, attenuates the development of pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 308: L208-L220, Terme JM, Lhermitte L, Asnafi V and Jalinot P. TGF-beta induces degradation of TAL1/SCL by the ubiquitin-proteasome pathway through AKT-mediated phosphorylation. Blood 113: , Thomas M, Docx C, Holmes AM, Beach S, Duggan N, England K, Leblanc C, Lebret C, Schindler F, Raza F, Walker C, Crosby A, Davies RJ, Morrell NW and Budd DC. Activin-like kinase 5 (ALK5) mediates abnormal proliferation of vascular smooth muscle cells from patients with familial pulmonary arterial hypertension and is involved in the 23

24 progression of experimental pulmonary arterial hypertension induced by monocrotaline. Am J Pathol 174: , Tuder RM. Pathology of pulmonary arterial hypertension. Semin Respir Crit Care Med 30: , Zaiman AL, Podowski M, Medicherla S, Gordy K, Xu F, Zhen L, Shimoda LA, Neptune E, Higgins L, Murphy A, Chakravarty S, Protter A, Sehgal PB, Champion HC and Tuder RM. Role of the TGF-beta/Alk5 signaling pathway in monocrotalineinduced pulmonary hypertension. Am J Respir Crit Care Med 177: , Ziino AJ, Ivanovska J, Belcastro R, Kantores C, Xu EZ, Lau M, McNamara PJ, Tanswell AK and Jankov RP. Effects of rho-kinase inhibition on pulmonary hypertension, lung growth, and structure in neonatal rats chronically exposed to hypoxia. Pediatr Res 67: , Figure Legends Figure 1. Inhibition of Akt attenuates PDGF-induced collagen synthesis and proliferation of PASMCs. (A) PASMCs with PDGF-BB (10 ng/ml) for h after which P-Akt-S473, P- Akt-T308 and total Akt were measured by Western blot. The image shown is representative immunoblots from 4 experiments. (B, C and D) PASMCs were incubated with PDGF-BB (10 ng/ml) in the absence and presence of triciribine (10 µm) for 24 h after which collagen-i protein level and cell proliferation were determined. (E, F and G) PASMCs were transfected with Akt sirna or control sirna and then incubated with PDGF-BB (10 ng/ml) for 24 h after which collagen-i protein level and cell proliferation were determined. (B) and (E) are representative immunoblots of collagen-i from 4 experiments. (C) and (F) are bar graph showing the changes in 24

25 protein levels of collagen-i quantified by scanning densitometry. (D) and (G) are bar graph showing the changes in cell proliferation. Results are expressed as mean ± SE; n = 4. P < 0.05 vs. control; P < 0.05 vs. PDGF only; P < 0.05 vs. control sirna. Figure 2. Inhibition of calpain-2 prevents PDGF-induced collagen synthesis and proliferation of PASMCs. (A, B and C) PASMCs were incubated with PDGF-BB (10 ng/ml) in the absence and presence of MDL28170 (20 µm) for 24 h after which collagen-i and cell proliferation were measured. (D, E and F) PASMCs were transfected with calpain-1 or calpain-2 sirna or control sirna and then incubated with PDGF-BB (10 ng/ml) for 24 h after which collagen-i and cell proliferation were measured. (A) and (D) are representative immunoblots of collagen-i. (B) and (D) are bar graph showing the changes in protein levels of collagen-i. (C) and (F) are bar graph showing the changes in cell proliferation. Results are expressed as mean ± SE; n = 4. P < 0.05 vs. control; P < 0.05 vs. PDGF only; P < 0.05 vs. control sirna. Figure 3. Inhibition of calpain attenuates Akt phosphorylation. (A and B) PASMCs were incubated with PDGF-BB (10 ng/ml) in the absence and presence of MDL28170 (20 µm) for 30 min after which P-Akt-S473, P-Akt-T308 and total Akt were measured by Western blot. (C and D) PASMCs were transfected with calpain-1 or calpain-2 sirna or control sirna and then incubated with PDGF-BB (10 ng/ml) for 30 min after which P-Akt-S473, P-Akt-T308 and total Akt were measured. (A) and (C) are representative immunoblots of P-Akt-S473, P-Akt-T308, and total Akt. (B) and (D) are bar graph showing the changes in protein levels of P-Akt-S473and P-Akt-T308. Results are expressed as mean ± SE; n = 4. P < 0.05 vs. control; P< 0.05 vs. PDGF only; P < 0.05 vs. control sirna; P < 0.05 vs. control sirna. 25

26 Figure 4. Overexpression of calpain-2 increases the levels of P-Akt-S473 and P-Akt-T308. PASMCs were transfected with plasmids containing human calpain-2 gene or empty plasmids for 24 h after which P-Akt-S473, P-Akt-T308 and total Akt were measured by Western blot. (A) is representative immunoblots of P-Akt-S473, P-Akt-T308, and total Akt. (B) is bar graph showing the changes in protein levels of P-Akt-S473and P-Akt-T308. Results are expressed as mean ± SE; n = 4. P < 0.05 vs. empty vector. Figure 5. Calpain knockout inhibits Akt phosphorylation in smooth muscle of pulmonary arteries in mice with chronic hypoxic PAH. 5 days after regimen of tamoxifen administration, control mice and ER-Cre+/-Capn4flox/flox mutant mice were exposed to room air (normoxia) or 10% oxygen (hypoxia) for 3 weeks. Lung slides from ER-Cre+/-Capn4flox/flox mutant and control mice exposed to normoxia or hypoxia were double-stained for α-actin (red) and P-Akt- S473, P-Akt-T308 or total Akt (green). (A) is representative images of 8 independent experiments. (B) is bar graph depicting the changes of P-Akt-S473 and P-Akt-T308 in smooth muscle of pulmonary arteries. Results are expressed as mean ± SE; n=8; P < 0.05 vs. normoxia in control mice; P < 0.05 vs. chronic hypoxia in control mice. Figure 6. Inhibition of TGFβ receptor prevents calpain-2-induced increases in the levels of P-Akt-S473 and P-Akt-T308. PASMCs were transfected with plasmids containing human calpain-2 gene or empty plasmids and then incubated with and without SB (10 µm) for 24 h after which P-Akt-S473, P-Akt-T308 and total Akt were measured by Western blot. (A) is representative immunoblots of P-Akt-S473, P-Akt-T308, and total Akt. (B) is bar graph showing the changes in protein levels of P-Akt-S473and P-Akt-T308. Results are expressed as mean ± SE; n = 5. P < 0.05 vs. empty vector in vehicle; P < 0.05 vs. empty vector in SB431542; P < 0.05 vs. calpain-2 vector in vehicle. 26

27 Figure 7. TGFβ neutralizing antibody does not affect PDGF-induced Akt phosphorylation. PASMCs were incubated with PDGF-BB (10 ng/ml) in the absence and presence of a cellimpermeable TGFβ neutralizing antibody (2 μg/ml) for 30 min after which protein levels of P- Akt-S473, P-Akt-T308, and total Akt collagen-i were measured. (A) is representative immunoblots from 4 experiments. (B) is bar graph showing the changes in protein levels of P- Akt-S473 and P-Akt-T308 quantified by scanning densitometry. Results are expressed as mean ± SE; n = 4. P < 0.05 vs. control; P < 0.05 vs. antibody only. Figure 8. TGFβ receptor inhibitor SB reduces PDGF-induced Akt phosphorylation, collagen synthesis and cell proliferation. PASMCs were incubated with PDGF-BB (10 ng/ml) in the absence and presence of SB (10 µm) for h after which protein levels of P- Akt-S473, P-Akt-T308, total Akt and collagen-i and cell proliferation were measured. (A) is representative immunoblots from 4 experiments. (B and C) are bar graph showing the changes in protein levels of P-Akt-S473, P-Akt-T308 and collagen-i quantified by scanning densitometry. (D) is bar graph showing the changes in cell proliferation. Results are expressed as mean ± SE; n = 4. P < 0.05 vs. control; P < 0.05 vs. PDGF. Figure 9. Knocking down ALK5 attenuates PDGF-induced collagen synthesis and cell proliferation. PASMCs were transfected with ALK5 sirna or control sirna and then incubated with PDGF-BB (10 ng/ml) for 24 h after which collagen-i protein level and cell proliferation were determined. (A) is representative immunoblots of collagen-i from 4 experiments. (B) is bar graph showing the changes in protein levels of collagen-i quantified by scanning densitometry. (C) is bar graph showing the changes in cell proliferation. Results are expressed as mean ± SE; n = 4. P < 0.05 vs. control; P < 0.05 vs. control sirna. 27

28 Figure 10. Knocking down Rictor attenuates PDGF-induced Akt phosphorylation in PASMCs. PASMCs were transfected with Rictor sirna or control sirna and then incubated with PDGF-BB (10 ng/ml) for 30 min after which protein levels of P-Akt-S473, P-Akt-T308, and total Akt were measured. (A) is representative immunoblots from 4 experiments. (B) is bar graph showing the changes in protein levels of P-Akt-S473 and P-Akt-T308. Results are expressed as mean ± SE; n = 4. P < 0.05 vs. control sirna; P < 0.05 vs. control sirna. Figure 11. Knocking down Rictor attenuates calpain-2-induced Akt phosphorylation in PASMCs. PASMCs were transfected with Rictor sirna or control sirna and then transfected plasmids containing human calpain-2 gene or empty plasmids for 24 h after which protein levels of P-Akt-S473, P-Akt-T308, and total Akt were measured. (A) is representative immunoblots from 3 experiments. (B) is bar graph showing the changes in protein levels of P-Akt-S473 and P- Akt-T308. Results are expressed as mean ± SE; n = 4. P < 0.05 vs. empty vector in control sirna; P < 0.05 vs. calpain-2 vector in control sirna. Figure 12. PI3 kinase inhibitor LY does not affect PDGF-induced calpain activation and calpain-2-induced Akt phosphorylation. (A) PASMCs were incubated with PDGF-BB (10 ng/ml) in the presence and absence of LY (50 µm) for 30 min after which calpain activity was measured as described in Materials and Methods. (B) and (C) PASMCs were transfected with plasmids containing human calpain-2 gene or empty plasmids and then incubated with and without LY (50 µm) for 24 h after which P-Akt-S473, P-Akt-T308 and total Akt were measured by Western blot. (B) is representative immunoblots of P-Akt-S473, P-Akt-T308, and total Akt. (C) is bar graph showing the changes in protein levels of P-Akt-S473and P-Akt-T

29 Results are expressed as mean ± SE; n = 3. P < 0.05 vs. control in vehicle; P < 0.05 vs. empty vector in vehicle; P < 0.05 vs. empty vector in LY Figure 13. A schematic pathway illustrating the role of calpain-2 in PDGF-induced Akt phosphorylation in PASMCs of pulmonary vascular remodeling. 29

30 A PDGF P-Akt-S473 P-Akt-T308 Total Akt Figure hours B GAPDH Collagen-I GAPDH E Akt Collagen-I GAPDH Triciribine Control PDGF Triciribine + PDGF Control Control Akt Akt sirna sirna sirna sirna 180 KD 37 KD C F 180 KD 37 KD Collagen-I level (Relative density to GAPDH) Collagen-I level (Relative density to GAPDH) Control PDGFTriciribinePDGF +Triciribine Control Control Akt Akt sirna sirna sirna sirna D G Proliferation (Absorbance) Proliferation (Absorbance) control PDGFTriciribinePDGF +Triciribine KD Control Control Akt Akt sirna sirna sirna sirna

31 Figure 2 A MDL MDL Control PDGF + PDGF Collagen-I 180 KD GAPDH Collagen-I level (Relative density to GAPDH) Control PDGF MDL MDL D E 1.0 F Control sirna Calpain-1 sirna Calpain-2 sirna PDGF Calpain-1 Calpain-2 Collagen-I GAPDH 37 KD B 80 KD 80 KD 180 KD 37 KD Collagen-I level (Relative density to GAPDH) Control Calpain-1 Calpain-2 sirna sirna sirna Control PDGF C Proliferation (Absorbance) Proliferation (Absorbance) control PDGF MDL MDL Control Calpain-1 Calpain-2 sirna sirna sirna Control PDGF

32 Figure 3 A P-Akt-S473 P-Akt-T308 Total Akt GAPDH C MDL28170 MDL28170 Control PDGF + PDGF Control sirna Calpain-1 sirna Calpain-2 sirna PDGF Calpain-1 Calpain-2 P-Akt-S473 P-Akt-T308 Total Akt GAPDH 37 KD 80 KD 80 KD 37 KD B Relative density to total Akt D Relative density to total Akt Control sirna Control PDGF MDL28170 MDL28170 Control sirna Calpain-1 sirna Calpain-1 sirna P-Akt-S473 P-Akt-T308 Calpain-2 sirna P-Akt-S473 P-Akt-T308 Calpain-2 sirna

33 Figure 4 A Overexpressed Native calpain-2 Empty vector Calpain-2 vector 83 KD 80 KD B P-Akt-S473 P-Akt-T308 Total Akt GAPDH P-Akt levels (Relative density to total Akt) P-Akt-S473 P-Akt-T308 Empty vector 37 KD Calpain-2 vector

34 A Normoxia S-473 T-308 Total-Akt Chronic hypoxia S-473 T-308 Total-Akt Figure 5 P-Akt α-actin Control mice Overlay P-Akt α-actin KO mice Overlay B P-Akt (Relative intensity to total-akt in SMC layer) P-S473 P-T308 normoxia chronic normoxia chronic hypoxia hypoxia control mice KO mice 100 μm