Yap/Taz mediates mTORC2-stimulated fibroblast activation and kidney fibrosis

Our previously published study demonstrated that mammalian target of rapamycin complex 2 (mTORC2) signaling mediates TGFβ1-induced fibroblast activation. However, the underlying mechanisms for mTORC2 in stimulating fibroblast activation remain poorly understood. Here, we found that TGFβ1 could stimulate mTORC2 and Yap/Taz activation in NRK-49F cells. Blocking either mTORC2 or Yap/Taz signaling diminished TGFβ1-induced fibroblast activation. In addition, blockade of mTORC2 could down-regulate the expression of Yap/Taz, connective tissue growth factor (CTGF), and ankyrin repeat domain 1 (ANKRD1). Overexpression of constitutively active Taz (Taz-S89A) could restore fibroblast activation suppressed by PP242, an mTOR kinase inhibitor in NRK-49F cells. In mouse kidneys with unilateral ureter obstructive (UUO) nephropathy, both mTORC2 and Yap/Taz were activated in the interstitial myofibroblasts. Ablation of Rictor in fibroblasts/pericytes or blockade of mTOR signaling with PP242 attenuated Yap/Taz activation and UUO nephropathy in mice. Together, this study uncovers that targeting mTORC2 retards fibroblast activation and kidney fibrosis through suppressing Yap/Taz activation.

Our previously published study demonstrated that mammalian target of rapamycin complex 2 (mTORC2) signaling mediates TGF␤1-induced fibroblast activation. However, the underlying mechanisms for mTORC2 in stimulating fibroblast activation remain poorly understood. Here, we found that TGF␤1 could stimulate mTORC2 and Yap/Taz activation in NRK-49F cells. Blocking either mTORC2 or Yap/Taz signaling diminished TGF␤1-induced fibroblast activation. In addition, blockade of mTORC2 could down-regulate the expression of Yap/Taz, connective tissue growth factor (CTGF), and ankyrin repeat domain 1 (ANKRD1). Overexpression of constitutively active Taz (Taz-S89A) could restore fibroblast activation suppressed by PP242, an mTOR kinase inhibitor in NRK-49F cells. In mouse kidneys with unilateral ureter obstructive (UUO) nephropathy, both mTORC2 and Yap/Taz were activated in the interstitial myofibroblasts. Ablation of Rictor in fibroblasts/ pericytes or blockade of mTOR signaling with PP242 attenuated Yap/Taz activation and UUO nephropathy in mice. Together, this study uncovers that targeting mTORC2 retards fibroblast activation and kidney fibrosis through suppressing Yap/Taz activation.
Chronic kidney disease, pathologically manifested as interstitial excessive extracellular matrix (ECM) 3 deposition and kidney fibrosis, is a leading cause for end-stage renal failure (1,2). Among all of the cell types, fibroblast is the major source for interstitial ECM production (3)(4)(5). To date, the mechanisms regulating fibroblast activation and kidney fibrosis remain to be further determined.
The Hippo pathway is an evolutionarily conserved kinase cascade that exerts crucial effects on regulating cell proliferation, organ size, and tissue regeneration (6). The core components of the Hippo pathway include Mst1/2, Sav1, Lats1/2, MOB1A, MOB1B, Yes-associated protein (Yap), and its paralogue transcriptional co-activator with PDZ-binding motif (Taz). Among them, Yap and Taz, classically activated by reducing Hippo pathway-mediated phosphorylation under certain stimuli, exhibit overlapping functions when expressed in the same cell (6 -9). After translocation into the nucleus, Yap/Taz interacts with several transcription factors, such as the TEA domain (TEAD) transcription factor family (TEAD1-4), ErbB4, and p73 to regulate target gene expression (10,11). In addition to the classic Hippo pathway, Yap/Taz may also be regulated by the Hippo-independent pathways (12,13). In diabetic nephropathy, activation of the EGFR-PI3K-Akt-CREB signaling pathway may up-regulate the expression of Yap and its target genes, including connective tissue growth factor (CTGF) and amphiregulin (14). Recently, Yap/Taz has been reported to be able to stimulate fibroblast activation and kidney fibrosis (15,16). However, the mechanisms for regulating Yap/ Taz activity during fibroblast activation and kidney fibrosis are not fully clarified.
The mammalian target of rapamycin (mTOR), an evolutionarily conserved member of the phosphatidylinositol-3-OH -kinase-related kinase family, plays an essential role in regulating cell growth, proliferation, and survival. As a core component, mTOR participates in forming two distinct complexes, named mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (17,18). MTORC2, composed of mTOR, Rictor, mSIN1, Protor-1, mLST8, and Deptor, plays a key role in modulating cell survival, metabolism, proliferation, and cytoskeleton homeostasis (19 -23). Our previously published study (24) demonstrated that mTORC2 signaling stimulates fibroblast activation and kidney fibrosis. Recently, accumulated evidence has unveiled interactions between mTORC2 and Hippo pathways. Sciarretta et al. (25) reported that Rictor/mTORC2 restrains the activity of MST1 kinase to preserve cardiac structure and function. Artinian et al. (26) found that mTORC2 can up-regulate the expression of Yap target genes through phosphorylating AMOTL2 at the site of Ser-760. Thus, we predicated that Rictor/mTORC2 may promote fibroblast activation and kidney fibrosis through stimulating Yap/Taz activation.
Here, we report that Rictor/mTORC2 signaling mediated TGF␤1-stimulated Yap/Taz up-regulation in kidney fibro-blasts. Blockade of mTORC2 signaling could largely reduce Yap/Taz activation and kidney fibrosis in mice with unilateral ureter obstructive (UUO) nephropathy.

Blockade of Rictor/mTORC2 signaling attenuates TGF␤1-induced Yap/Taz up-regulation
To investigate the role for mTORC2 signaling in Yap/Taz activation in kidney fibroblasts, we generated primary cultured mouse kidney fibroblasts from Rictor fl/fl mice and infected the cells with adenovirus carrying Cre recombinase for 48 h to induce Rictor gene deletion (Fig. S1). The fibroblasts were stimulated with TGF␤1 (2 ng/ml) for different durations as indicated. A Western blot assay revealed that TGF␤1 could remarkably up-regulate Yap/Taz expression in a time-dependent manner in the control fibroblasts, whereas ablation of Rictor largely prohibited their induction (Fig. 1A). We also treated NRK-49F cells, a rat kidney interstitial fibroblast cell line, with the mTOR inhibitor PP242 and mTORC1 inhibitor rapamycin, respectively. The results showed that PP242 at 16 nM could reduce TGF␤1-induced Akt and S6 phosphorylation by about 34 and 39%, respectively (Fig. 1B). PP242 but not rapamycin could markedly inhibit TGF␤1-induced Yap/Taz up-regulation (Fig. 1C). PP242 treatment could also decrease the mRNA abundance for Yap/Taz as well as its target genes, including CTGF and ankyrin repeat domain 1 (ANKRD1), in NRK-49F cells (Fig. 1D), whereas rapamycin could not (Fig. 1E). The results suggest that mTORC2 is required for TGF␤1-induced Yap/Taz activation in NRK-49F cells.
To further decipher the role of Akt, the major downstream molecule for mTORC2 signaling, in regulating Yap/Taz activity, we treated NRK-49F cells with Akt1/2 inhibitor, followed by TGF␤1 treatment. The results showed that blocking Akt could largely reduce the up-regulation of Yap/Taz, CTGF, and ANKRD1, respectively (Fig. 1, F and G). We also treated NRK-49F cells with cycloheximide (CHX) and actinomycin D to inhibit gene translational and transcriptional activity, respectively. The Western blot assay showed that CHX and actinomycin D could largely prohibit TGF␤1-induced Yap/Taz up-regulation ( Fig. 1, H and I). Taken together, it may be concluded that Rictor/mTORC2/Akt signaling is required for TGF␤1-induced Yap/Taz activation in kidney fibroblasts.

Yap/Taz induction mediates mTORC2-stimulated kidney fibroblast activation
Our previous study demonstrated that Rictor/mTORC2 signaling mediates TGF␤1-induced fibroblast activation (24). Consistent with that report, we found that ablation of the Rictor gene in primary cultured kidney fibroblasts could largely attenuate TGF␤1-induced fibroblast activation (Fig. 2, A and B). Blockade of mTOR signaling with PP242 could also markedly inhibit TGF␤1-induced FN and ␣-SMA protein expression in NRK-49F cells (Fig. 2, C and D). In addition, blocking Yap/Taz with verteporfin markedly inhibited TGF␤1-induced NRK-49F cell activation (Fig. 2, E and F). The Western blot assay showed that verteporfin could not affect TGF␤1-stimulated Smad3 phosphorylation (Fig. 2E, right). NRK-49F cells were also transfected with Yap/Taz siRNAs, respectively. The Western blot assay showed that knocking down Yap/Taz expression could inhibit TGF␤1-induced fibroblast activation (Fig. 2, G-J). To further decipher the role for Yap/Taz induction in mediating Rictor/mTORC2-stimulated fibroblast activation, we transfected NRK-49F cells with plasmid carrying a constitutively active form of Taz (Taz-S89A) (27) (Fig. 2K). The results showed that expression of Taz-S89A could largely restore TGF␤1-induced fibroblast activation, which was suppressed by PP242 treatment (Fig. 2, L and M). Taken together, these data demonstrated that Yap/Taz induction mediates mTORC2stimulated kidney fibroblast activation.

Both mTORC2 and Yap/Taz signaling are activated in myofibroblasts from the fibrotic kidneys with UUO nephropathy
The aforementioned data demonstrated that Yap/Taz mediates mTORC2-stimulated fibroblast activation in cultured kidney fibroblasts. To further decipher the interaction between mTORC2 and Yap/Taz signaling in myofibroblasts within the fibrotic kidneys, we generated UUO model and examined the abundance of these two signaling pathway proteins in the fibrotic kidneys in mice. Pronounced induction of p-Akt (Ser-473), p-Yap (Ser-127), Yap/Taz, and TEAD as well as the reduction of LATS1 were detected in the kidneys at days 3, 7, and 14 after UUO (Fig. 3A). The mRNA abundance for Yap/Taz, TEAD1-4, CTGF, and ANKRD1 was increased in the UUO kidneys (Fig. 3B). To explore whether both mTORC2 and Yap/ Taz were activated in myofibroblasts from the UUO kidneys, we co-stained the UUO kidney tissue with antibodies against ␣-SMA and p-Akt (Ser-473), Yap/Taz, and TEAD, respectively. The results showed that p-Akt (Ser-473), Yap/Taz, and TEAD could be detected in myofibroblasts from the UUO kidneys. It is of note that p-Akt (Ser-473), Yap/Taz and TEAD were also detected in epithelial cells after UUO (Fig. 3C). Together, these data showed that mTORC2 and Yap/Taz are co-activated in the myofibroblasts from the fibrotic kidneys in mice with UUO nephropathy.

Ablation of Rictor in fibroblasts diminishes Yap/Taz activation and UUO nephropathy in mice
To further investigate the role of Rictor/mTORC2 signaling in regulating Yap/Taz and fibroblast activation, we generated a mouse model with inducible ablation of Rictor in fibroblast by breeding Rictor-floxed mice with Gli1-CreERT2 transgenic mice (28 -30). After breeding several times (Fig. S2A), we obtained mice with genotyping Gli1-Cre ϩ/Ϫ , Rictor fl/fl (Fig.  S2B, lane 3). Mice with genotyping Gli1-Cre ϩ/Ϫ , Rictor fl/fl were injected intraperitoneally with tamoxifen for 5 consecutive days. The same-sex littermates with genotyping Gli1-Cre Ϫ/Ϫ , Rictor fl/fl were injected with tamoxifen and treated as control littermates. Two days after the last injection, both control littermates and knockouts were subjected to UUO (Fig. 2C). The protein abundance for Yap/Taz, FN, and ␣-SMA was remarkably increased in the UUO kidneys from control littermates compared with the sham kidneys, which was largely decreased in the UUO kidneys from the knockouts (Fig. 4, A and B). Immune staining further confirmed the reduction of Yap/Taz in myofibroblasts from the knockouts after UUO compared

MTORC2 promotes Yap/Taz activation and kidney fibrosis
with those from the control littermates (Fig. 4C). The mRNA abundance for Yap/Taz, TEAD1-4, CTGF, and ANKRD was also decreased in the UUO kidneys from the knockouts com-pared with control littermates (Fig. 4D). These results suggest that ablation of Rictor in fibroblasts could reduce Yap/Taz and fibroblast activation in mice with UUO nephropathy.

MTORC2 promotes Yap/Taz activation and kidney fibrosis
We stained the kidney sections with periodic acid-Schiff, Masson, and Sirius red. In the UUO kidneys from the control littermates, remarkable tubular atrophy and interstitial extracellular matrix deposition could be detected, which were largely alleviated in the knockout kidneys (Fig. 4E). The fibrotic area and total collagen content within the UUO kidneys were also significantly decreased in the knockouts compared with those from the control littermates (Fig. 4, F and G). Taken together, these data suggest that ablation of Rictor in fibroblasts reduces Yap/Taz induction and kidney fibrosis in mice with UUO nephropathy.

Blockade of mTORC2 signaling with PP242 attenuates Yap/Taz induction and UUO nephropathy in mice
In cultured NRK-49F cells, we found that blocking mTORC2 with PP242 could inhibit TGF␤1-induced Yap/Taz signaling activation. We then wanted to know whether PP242 could inhibit Yap/Taz activation and kidney fibrosis in mice with

MTORC2 promotes Yap/Taz activation and kidney fibrosis
UUO nephropathy. PP242 was administered (20 mg/kg⅐day) intraperitoneally from day 2 before to days 7 and 14 after UUO in male CD1 mice. In the UUO kidneys, the protein abundance for p-Akt (Ser-473) and Yap/Taz was largely reduced in mice with PP242 administration compared with the vehicle control (Fig. 5, A and B). The mRNA abundance of Yap/Taz, CTGF, ANKRD1, and TEAD1-4 was also reduced in the UUO kidneys after PP242 administration compared with those treated with vehicle alone (Fig. 5C). Immunofluorescent staining revealed that PP242 could markedly reduce Yap/Taz induction in the myofibroblasts from the fibrotic kidneys (Fig. 5D). These data suggest that suppressing mTORC2 with PP242 could inhibit Yap/Taz activation in myofibroblasts from the UUO kidneys in mice.
We then examined kidney fibrosis in mice after PP242 treatment. The results showed that PP242 could reduce FN and ␣-SMA protein abundance in the UUO kidneys compared with those treated with vehicle alone (Fig. 6A). Immunostaining for FN and ␣-SMA further confirmed the results of a Western blot assay (Fig. 6B). Periodic acid-Schiff, Masson, and Sirius red staining showed that tubule injury as well as interstitial matrix deposition were largely attenuated in mice treated with PP242 (Fig. 6B). PP242 could also remarkably decrease fibrotic area and total collagen content in the kidneys at day 14 after UUO (Fig. 6, C and D).
To evaluate the inflammatory cell accumulation in the UUO kidneys, we stained kidney tissue with anti-Ly6b and anti-F4/80 to identify neutrophil and macrophage, respectively. The

MTORC2 promotes Yap/Taz activation and kidney fibrosis
results showed that neutrophil as well as macrophage accumulation was increased in the UUO kidneys from mice treated with vehicle alone, and their accumulation was much lower in PP242-treated UUO kidneys (Fig. 6, B, E, and F). Therefore, it may be concluded that PP242 could inactivate Yap/Taz and ameliorate UUO nephropathy in mice.

Discussion
It has been reported that the mTORC2 pathway is important for promoting the fibrosis process (24,(31)(32)(33). In addition, Yap/Taz is the mechano-transducer that regulates TGF␤1 signaling and renal fibrogenesis (15,16,27,34). In this study, we demonstrated that Yap/Taz is a critical mediator for Rictor/ mTORC2 in stimulating fibroblast activation and kidney fibrosis based on several lines of evidence. First, both mTORC2 and Yap/Taz were activated in the TGF␤1-stimulated kidney fibroblasts and myofibroblasts from the UUO kidneys. Second, ablation of Rictor in fibroblasts/pericytes or pharmacological inhibition of mTORC2 signaling could attenuate Yap/Taz activation and ameliorate kidney fibroblast activation and obstructive nephropathy in mice. Third, overexpression of constitutively active Taz could restore fibroblast activation, which was suppressed by mTORC2 signaling inhibition. To the best of our knowledge, this is the first report that demonstrates the crucial role of Yap/Taz activation in mediating Rictor/ mTORC2-promoted kidney fibroblast activation and kidney fibrosis.
The mTOR and Hippo pathways are two major signaling cascades that coordinately regulate cell growth and proliferation (35). Substantial evidence has demonstrated that cross-talk exists between them (25,26,36,37). Artinian et al. (26) found that mTORC2 phosphorylating AMOTL2 at Ser-760 results in the blockade of its YAP-binding properties and induces the up-regulation of Yap target genes. MTORC2 may also phosphorylate MST1 at Ser-438 and reduce its dimerization and activity, thereby reducing the phosphorylation of Yap and lead-

MTORC2 promotes Yap/Taz activation and kidney fibrosis
ing to Yap activation (25). Additionally, mTOR has been reported to be able to up-regulate Yap expression through impairing the autophagy-induced Yap degradation (36). Yap could regulate the mTOR pathway via miR-29 (37). Studies have disclosed that activated LATS1/2 could phosphorylate Yap/Taz, thereby inducing their cytoplasmic sequestration and inactivation (7, 38 -40). In this study, we demonstrated that TGF␤1 could up-regulate Yap/Taz expression through mTORC2 signaling-stimulated Yap/Taz transcriptional activation. This conclusion is supported by the following evidence: 1) TGF␤1 could markedly up-regulate the protein and mRNA expression of Yap/Taz in cultured kidney fibroblasts, which were largely suppressed by PP242 or Akt1/2 inhibitor; 2) in the fibrotic kidneys with UUO nephropathy, the mRNA and protein abundance of Yap/Taz and TEAD1-4 were increased, and these were largely suppressed in the mice with fibroblast ablation of Rictor or treated with PP242; 3) blocking gene transcription and translation with actinomycin D and cycloheximide, respectively, could almost completely abolish TGF␤1induced Yap/Taz up-regulation. However, the molecular mechanisms for Rictor/mTORC2 signaling in stimulating the Yap/Taz transcription remain to be further clarified.

MTORC2 promotes Yap/Taz activation and kidney fibrosis
vious study reported that ablation of Rictor in fibroblasts could attenuate UUO nephropathy in mice (24). In that study, S100A4-Cre transgenic mice were employed to generate the mice with fibroblast ablation of the Rictor. Recently, studies have demonstrated that Gli1 marks a perivascular mesenchymal stem cell-like progenitor population (28,44). In kidneys with UUO nephropathy, resident Gli1 ϩ cells are able to be transformed to myofibroblasts and promote kidney fibrosis (28,44,46). In this study, we generated a mice model with Gli1 ϩfibroblast/pericyte ablation of Rictor by cross-breeding Rictorfloxed mice with the Gli1-CreERT2 transgenic mice, in which Cre recombinase expression is under the control of the endogenous Gli1 promoter (46). Compared with the S100A4-Cre transgenic mice, Gli1-CreERT2 transgenic mice provide an alternative option that deletes the target genes in fibroblasts/ pericytes. Additionally, Cre recombinase expression in Gli1-CreERT2 transgenic mice is tamoxifen-inducible, which may facilitate the investigation for the pathogenic role of target genes under diseased conditions. Consistent with our previous report using S100A4-Cre transgenic mice (24), kidney fibrosis was attenuated in the UUO kidneys from the mice with Gli1 ϩfibroblast/pericyte ablation of Rictor. It should be noted that only part of the fibroblasts within the kidneys express Gli1, so the effects of Rictor/mTORC2 signaling in promoting fibroblast activation and kidney fibrosis may be underscored by using this model. Nevertheless, based on the results from two different mouse models with fibroblast ablation of Rictor, it is concluded that Rictor/mTORC2 signaling activation plays a crucial role in promoting fibroblast activation and kidney fibrosis. This conclusion was further supported by the pharmacologic inhibition of mTORC2 signaling with PP242, in which kidney fibrosis induced after UUO operation was largely ameliorated. It should be noted that Yap/Taz was also activated in tubular epithelial cells after UUO, suggesting a potential role for epithelial Hippo pathway in kidney fibrosis.
In summary, our study demonstrated that Yap/Taz is indispensable for Rictor/mTORC2-stimulated kidney fibrogenic process. Blockade of mTORC2/Yap/Taz may shed new light on attenuating kidney fibrosis for patients with chronic kidney disease.
Male CD-1 mice weighing ϳ18 -20 g were acquired from the specific pathogen-free Laboratory Animal Center of Nanjing Medical University, and we performed UUO experiments on these as reported previously (47). The animals were divided into two groups; mice were 1) injected with vehicle or 2) treated with PP242 (20 mg/kg⅐day) from 2 days before to 1 or 2 weeks after surgery. PP242 (catalog no. S221, Selleckchem) was prepared in a 10% DMSO solution and injected intraperitoneally. The mice were euthanized on days 7 and 14 after UUO. The UUO kidneys as well as the contralateral kidneys were removed. One portion of the kidney was fixed in 10% phosphate-buffered formalin, followed by paraffin embedding for histological and immunohistochemical staining. Another portion was immediately frozen in Tissue-Tek optimum cutting temperature compound (Sakura Finetek, Torrance, CA) for cryosection. The remaining kidney tissue was snap-frozen in liquid nitrogen and stored at Ϫ80°C for extraction of RNA and protein.
All animals were maintained in the specific pathogen-free Laboratory Animal Center of Nanjing Medical University according to the guidelines of the institutional animal care and use committee at Nanjing Medical University (Nanjing, China). All experiments were performed in accordance with the approved guidelines and regulations of the Animal Experimentation Ethics Committee at Nanjing Medical University. The work described has been carried out in accordance with EU Directive 2010/63/EU for animal experiments.

Histology and immunohistochemistry
Kidney samples were fixed in 10% neutral formalin and embedded in paraffin. Sections at 3-m thickness were stained with periodic acid-Schiff, Masson, and Sirius red. For immunohistochemical staining, paraffin-embedded kidney sections were deparaffinized, hydrated, and antigen-retrieved. Endogenous peroxidase activity was quenched by 3% H 2 O 2 . Sections were then blocked with 10% normal donkey serum, followed by incubation with anti-Ly6b (catalog no. 0715, Bio-Rad) overnight at 4°C. After incubation with secondary antibody for 1 h, sections were incubated with ABC reagents for 1 h at room temperature before being subjected to substrate 3-amino-9ethylcarbazole or 3,3Ј-diaminobenzidine (Vector Laboratories, Burlingame, CA). Slides were viewed with a Nikon Eclipse 80i microscope equipped with a digital camera (DS-Ri1, Nikon, Shanghai, China).

Western blot analysis
NRK-49F cells and primary cultured kidney fibroblasts were lysed in 1ϫ SDS sample buffer. The kidney tissues were lysed with radioimmunoprecipitation assay solution containing 1% Nonidet P-40, 0.1% SDS, 100 g/ml phenylmethylsulfonyl fluoride, 1% protease inhibitor mixture, and 1% phosphatase I and II inhibitor mixture (Sigma) on ice. The supernatants were collected after centrifugation at 13,000 ϫ g at 4°C for 30 min.
Protein concentration was determined by a bicinchoninic acid protein assay (BCA kit; Pierce) according to the manufacturer's instructions. An equal amount of protein was loaded onto SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The primary antibodies were as follows: anti-GAPDH (catalog no. FL-335, Santa Cruz Biotechnology, Inc., Dallas, TX), anti-p-Akt (Ser-473) (catalog no. 3868, Cell Signaling Technology), anti-FN (catalog no. F3648, Sigma-Aldrich), anti-␣-SMA (catalog no. A5228, Sigma-Aldrich), anti-TEAD (D3F7L) (catalog no. 13295, Cell Signaling Technology), anti-Yap/Taz (D24E4) (catalog no. 8418, Cell Signaling Technology), and anti-Yap (catalog no. 4912S, Cell Signaling Technology). Quantification was performed by measuring the intensity of the signals with the aid of the National Institutes of Health ImageJ software package.

RNA isolation and real-time quantitative reverse transcription-PCR
Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized with 1 g of total RNA, ReverTra Ace (Vazyme, Nanjing, China), and oligo(dT) 12-18 primers. Gene expression was measured by real-time PCR assay (Vazyme) and a 7300 real-time PCR system (Applied Biosystems, Foster City, CA). The amount of mRNA or gene relative to internal control was calculated using the equation, 2⌬CT, in which ⌬CT ϭ CT gene Ϫ CT control .

Statistical analysis
All data examined are presented as mean Ϯ S.E. Statistical analysis of the data was performed using the SigmaStat software (Jandel Scientific Software, San Rafael, CA). Comparison between groups was made using one-way analysis of variance, followed by the Student-Newman-Keuls test. p Ͻ 0.05 was considered as statistically significant.