The immunophilin ligands cyclosporin A and FK506 suppress prostate cancer cell growth by androgen receptor-dependent and -independent mechanisms

The androgen receptor (AR) contributes to growth of prostate cancer even under conditions of androgen ablation. Thus, new strategies to target AR activity are needed. The AR interacts with the immunophilin FK506-binding protein 52 (FKBP52), and studies in the FKBP52 knockout mouse have shown that this protein is essential to AR activity in the prostate. Therefore, we tested whether the immunophilin ligand FK506 affected AR activity in prostate cancer cell lines. We also tested the hypothesis that the AR interacts with another immunophilin, cyclophilin 40 (Cyp40), and is regulated by its cognate ligand cyclosporin A (CsA). We show that levels of FKBP52, FKBP51, Cyp40, and a related co-chaperone PP5 were much higher in prostate cancer cells lines [(LNCaP), PC-3, and DU145] compared with primary prostate cells, and that the AR of LNCaP cells can interact with Cyp40. In the absence of androgen, CsA caused inhibition of cell growth in the AR-positive LNCaP and AR-negative PC-3 and DU145 cell lines. Interestingly, FK506 only inhibited LNCaP cells, suggesting a dependence on the AR for this effect. Both CsA and FK506 inhibited growth without inducing apoptosis. In LNCaP cells, CsA completely blocked androgen-stimulated growth, whereas FK506 was partially effective. Further studies in LNCaP cells revealed that CsA and FK506 were able to block or attenuate several stages of AR signaling, including hormone binding, nuclear translocation, and activity at several AR-responsive reporter and endogenous genes. These findings provide the first evidence that CsA and FK506 can negatively modulate proliferation of prostate cells in vitro. Immunophilins may now serve as new targets to disrupt AR-mediated prostate cancer growth.     

Use of tacrolimus, a potent antifibrotic agent, in bleomycin-induced lung fibrosis.

Idiopathic pulmonary fibrosis has a poor prognosis and few efficacious treatments. The immunosuppressant cyclosporin A has been shown to inhibit tumour growth factor (TGF)-beta-induced collagen deposition in vitro, and is widely used in Japan as a potent antifibrotic agent. Tacrolimus (FK506) is another attractive immunosuppressant, which may be useful in the treatment of pulmonary fibrosis. The aim of the present study was to elucidate the antifibrotic effect of FK506. The inhibitory effect of FK506 on collagen synthesis in cultured lung fibroblastic cells, TIG-3-20, and its antifibrotic effect on bleomycin (BLM)-induced pulmonary fibrosis in mice was investigated. FK506 inhibited TGF-beta-induced collagen synthesis, and suppressed the expression of TGF-beta type I receptor (TbetaR-I) in TIG-3-20 cells. Consistent with the in vitro findings, FK506 treatment starting on day 6 attenuated BLM-induced pulmonary fibrosis, in part, via reduced TbetaR-I expression. FK506 treatment in the acute BLM injury phase unexpectedly increased pro-inflammatory cytokine levels in bronchoalveolar lavage fluid and enhanced lung injury, resulting in poor survival. In conclusion, the present results suggest that FK506 has a potent antifibrotic effect and may be useful for the treatment of pulmonary fibrosis, although its use in the acute inflammatory phase may exacerbate lung injury.     

Effects of cyclosporin A and FK506 on Fc epsilon receptor type I-initiated increases in cytokine mRNA in mouse bone marrow-derived progenitor mast cells: resistance to FK506 is associated with a deficiency in FK506-binding protein FKBP12.

The inhibitory effects of cyclosporin A (CsA) and FK506 on Fc epsilon receptor type I-initiated increases in cytokine mRNA and the expression of their intracellular binding proteins were studied in interleukin 3 (IL-3)-dependent, mouse bone marrow-derived mast cells (BMMCs). In BMMCs sensitized with IgE anti-trinitrophenyl, CsA inhibited trinitrophenylated bovine serum albumin-induced increases in mRNA for IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 in a dose-related manner (IC50 values of 4, 65, and 130 nM, respectively). FK506 did not inhibit hapten-specific increases of mRNA for TNF-alpha or IL-6, and for IL-1 beta the IC50 was greater than 50-fold higher than that of CsA. Neither agent inhibited exocytosis of the endogenous secretory granule mediators beta-hexosaminidase and histamine at the IC50 values for inhibition of increases in cytokine mRNA. BMMCs expressed cyclophilin, and CsA inhibited the phosphatase activity of cellular calcineurin with an IC50 of approximately 8 nM. That CsA inhibited IL-1 beta mRNA accumulation in IgE-activated BMMCs with an IC50 similar to that for inhibition of calcineurin activity, whereas the IC50 values were approximately 20-fold higher for the inhibition of TNF-alpha and IL-6 mRNA, suggests that the induction of TNF-alpha and IL-6 is less dependent upon calcineurin activity than is the induction of IL-1 beta. BMMCs were deficient in the 12-kDa FK506-binding protein FKBP12, but not FKBP13, as assessed by RNA and protein blot analyses. FK506 did not inhibit calcineurin phosphatase activity in BMMCs, even at drug concentrations of 1000 nM. The resistance of BMMCs to inhibition of Fc epsilon receptor type I-mediated increases in cytokine mRNA by FK506 is most likely due to their deficiency of FKBP12 and the related inability to inhibit the activity of calcineurin.     

FK506 can activate transforming growth factor-beta signalling in vascular smooth muscle cells and promote proliferation

AIMS: FK506-binding protein (FKBP) 12 is an inhibitor of transforming growth factor (TGF)-beta type I receptors. Several lines of evidence support the view that TGF-beta stimulates vascular smooth muscle cell (VSMC) proliferation and matrix accumulation. We investigated the effect of FK506, also known as tacrolimus, on cellular proliferation and on matrix protein production in human VSMCs. METHODS AND RESULTS: We measured cell proliferation with flow cytometry using BrdU incorporation and fluorimetrically by measuring DNA concentration with Hoechst 33258. Western blot assay of whole-cell lysates was used to measure the levels of signalling proteins involved in proliferative pathways, in particular beta-catenin, pErk, pAkt, pmTOR, and cyclin D1. Collagen synthesis was also investigated by Western blotting. The TGF-beta signal was studied by both Western blotting and confocal microscopy. We used the SiRNA technique for FKBP12 gene silencing. Our results show that FK506 stimulates VSMC proliferation and collagen type I production. FK506 enhanced beta-catenin levels and activated the extracellular signal-regulated kinase, Akt, and mammalian target of rapamycin kinase, which are important effectors of proliferation. Accordingly, cyclin D1 expression was increased. We also demonstrate that FK506 activates the TGF-beta signal in VSMCs and that, through this mechanism, it stimulates cell proliferation. CONCLUSION: FK506 can act as a growth factor for VSMCs.     

Effect of ligand of peroxisome proliferator-activated receptor γ on the biological characters of hepatic stellate cells

AIM: To study the effect of rosiglitazone, which is a ligand of peroxisome proliferator-activated receptor gamma (PPARγ), on the expression of PPARγ in hepatic stellate cells (HSCs) and on the biological characteristics of HSCs.METHODS: The activated HSCs were divided into three groups: control group, 3 μmol/L rosiglitazone group, and 10 μmol/L rosiglitazone group. The expression of PPARγ,α-smooth muscle actin (α-SMA), and type Ⅰ and Ⅲ collagen was detected by RT-PCR, Western blot and immunocytochemical staining, respectively. Cell proliferation was determined with methylthiazolyltetrazolium (MTT) colorimetric assay. Cell apoptosis was demonstrated with flow cytometry.RESULTS: The expression of PPARγ at mRNA and protein level markedly increased in HSCs of 10 μmol/L rosiglitazone group (tvalue was 10.870 and 4.627 respectively, P<0.01in both). The proliferation of HSCs in 10 μmol/L rosiglitazone group decreased significantly (t = 5.542, P<0.01), α-SMA expression level and type Ⅰ collagen synthesis ability were also reduced vs controls (tvalue= 10.256 and 14.627respectively, P<0.01 in both). The apoptotic rate of HSCs significantly increased in 10 μmol/L rosiglitazone group vs control (x2= 16.682, P<0.01).CONCLUSION: By increasing expression of PPARγ in activated HSCs, rosiglitazone, an agonist of PPARγ,decreases α-SMA expression and type Ⅰ collagen synthesis,inhibits cell proliferation, and induces cell apoptosis.     

Effect of ligand of peroxisome proliferator-activated receptor γ on the biological characters of hepatic stellate cells

AIM: To study the effect of rosiglitazone, which is a ligand of peroxisome proliferator-activated receptor gamma (PPARγ), on the expression of PPARγ in hepatic stellate cells (HSCs) and on the biological characteristics of HSCs.METHODS: The activated HSCs were divided into three groups: control group, 3 μmol/L rosiglitazone group, and 10 μmol/L rosiglitazone group. The expression of PPARγ,α-smooth muscle actin (α-SMA), and type Ⅰ and Ⅲ collagen was detected by RT-PCR, Western blot and immunocytochemical staining, respectively. Cell proliferation was determined with methylthiazolyltetrazolium (MTT) colorimetric assay. Cell apoptosis was demonstrated with flow cytometry.RESULTS: The expression of PPARγ at mRNA and protein level markedly increased in HSCs of 10 μmol/L rosiglitazone group (tvalue was 10.870 and 4.627 respectively, P<0.01in both). The proliferation of HSCs in 10 μmol/L rosiglitazone group decreased significantly (t = 5.542, P<0.01), α-SMA expression level and type Ⅰ collagen synthesis ability were also reduced vs controls (tvalue= 10.256 and 14.627respectively, P<0.01 in both). The apoptotic rate of HSCs significantly increased in 10 μmol/L rosiglitazone group vs control (x2= 16.682, P<0.01).CONCLUSION: By increasing expression of PPARγ in activated HSCs, rosiglitazone, an agonist of PPARγ,decreases α-SMA expression and type Ⅰ collagen synthesis,inhibits cell proliferation, and induces cell apoptosis.     

肾移植术后抗排斥药FK506的临床应用

目的研究FK506预防肾移植术后排斥反应的效果和安全性。方法肾移植患者22例,其中18例为始用组,4例为切换组。FK506起始用0.2me／(kg·d),以后逐步减量,3个月后维持血浓度于3～12μg/L水平。切换组于停用CsA24h后应用FK506,剂量和血浓度与始用组相同。同时合并应用MMF0.5g,每日3次口服,以及术后前10天大剂量甲基强的松龙静滴,第11天改强的松口服并减量,6个月后维持强的松15mg／d。所有病例均严密观察并行血尿等生化分析。结果始用组移植肾功能好,平均血肌酐水平l02μmol／L,无一例出现排斥反应。切换组中2例异常的肝功能好转;肾功能进行性减退的2例切换后,血肌酐相对稳定。有血糖升高4例和高血压5例,用药后能控制,其他副反应有上呼吸道和下尿路感染、胸痛、恶心、呕吐、腹泻、腹部不适等。结论FK506是肾移植术后有确切疗效的基础抗排斥药,与MMF、皮质醇合用能有效地预防急性排斥的发生,并可控制慢性排斥的进展。应用剂量适当,无明显的肝、肾毒副作用,但有血糖升高及高血压副作用,药物可以控制。其它呼吸道、尿路、消化道和神经系统副反应轻,不妨碍临床用药。     

血小板衍化生长因子对血管平滑肌细胞增殖及胶原蛋白合成的影响

目的 :观察血小板衍化生长因子 （PDGF）对培养的血管平滑肌细胞 （VSMC）增殖及胶原蛋白合成的影响。方法 :采用培养的兔动脉 VSMC,应用 3H- Td R的 3H-脯氨酸掺入方法 ,观察 PDGF- BB对兔 VSMC DNA合成以及胶原蛋白合成的影响。结果 :PDGF- BB可促进处于静止状态的兔 VSMC DNA及胶原蛋白的合成 ,并呈现明显的浓度依赖关系 ,在 40 μg/ L 的浓度时 DNA及胶原蛋白的合成达到高峰 ,DNA及胶原蛋白分别处于 36 h和 48h合成最为显著。结论 :PDGF- BB可明显促进培养的 VSMC增殖及胶原蛋白的合成。     

The role of protein kinases and phosphatases in erythroid colony formation of elderly

Using H-7, HA1001, FK506, cyclosporin A (CsA) and okadaic acid (OA), which are protein kinase and phosphatase inhibitors, we examined qualitative changes in hematopoietic precursor cells due to aging from the viewpoint of the role of protein kinases and phosphatases. Though H-7 and OA suppressed erythroid colony formation both in the elderly (age: 72-92, median: 86) and the young (age: 22-39, median: 29), no change due to aging was noted. HA1001 did not affect erythroid colony formation either in the elderly or the young. Erythroid colony formation was enhanced by FK506 and CsA in the young, however, erythroid colony formation was suppressed in the elderly. Similar examinations using cell fractions of non-T, non-macrophage, non-T + T, and CD34 positive cells were performed in both groups. Enhancement of erythroid colony formation in the young and suppression in the elderly by FK506 using unseparated MNC disappeared after removal of T cells. Enhancement of colony formation in the young and suppression of colony formation in the elderly were recovered when T cells were added again. The effects of FK506 and CsA on erythroid colony formation were thought to be the results of T cell inactivation, and the different sensitivity to FK506 and CsA in the elderly and young seemed to be the result of changes in the control mechanisms of hematopoiesis, such as the regulation of cytokine production by T cells, caused by aging.     

Differential effects between tauroursodeoxycholic and taurochenodeoxycholic acids in hepatic fibrosis: An assessment by primary cultured Ito and Kupffer cells from the rat liver*

The pathogenesis of hepatic fibrosis in cholestasis is still unknown, except for endotoxaemia. There is a possibility that the elevation of serum bile acids in cholestasis may play an important role in hepatic fibrogenesis due to a reaction to perisinusoidal cells, such as Ito or Kupffer cells. To assess the effects of bile acids, we investigated the cell proliferation and collagen formation of primary cultured Ito cells that were incubated with a Kupffer cell conditioned medium (KCCM) treated with either taurochenodeoxycholic acid (TCDCA) or tauroursodeoxycholic acid (TUDCA) in short-term (8 h) or long-term (48 h) cultures. KCCM treated with TCDCA (100 μmol/L) but not with TUDCA increased cell proliferation of Ito cells in short-term cultures and also partially elevated collagen formation by Ito cells in long-term cultures. The release of tumour necrosis factor-α (TNFα) from Kupffer cells was increased by TCDCA in short-term cultures, but not in long-term cultures. The release of transforming growth factor-β₁ (TGFβ₁) from Kupffer cells was increased by TCDCA in long-term cultures, but not in the short-term cultures. TUDCA showed no significant effect on the release of TNFα and TGFβ₁ from Kupffer cells. TUDCA or TCDCA itself showed no direct effect on the cell proliferation and collagen formation of Ito cells. In conclusion, these findings are thus considered to show the potentially important role of TCDCA on the development of hepatic fibrosis in the early phase of cholestasis without endotoxaemia.     

Immunosuppressive effects of tautomycetin in vivo and in vitro via T cell-specific apoptosis induction

Tautomycetin (TMC) was identified as an immunosuppressor of activated T cells. Inhibition of T cell proliferation with TMC was observed at concentrations 100-fold lower than those needed to achieve maximal inhibition with cyclosporin A (CsA). TMC specifically blocked tyrosine phosphorylation of intracellular signal mediators downstream of Src tyrosine kinases in a T cell-specific manner, leading to apoptosis due to cleavage of Bcl-2, caspase-9, caspase-3, and poly(ADP-ribose) polymerase, but not caspase-1. In TMC-treated rats that received a heterotopic cardiac allograft, the graft survived more than 160 days, comparable to graft survival in allografted rats treated with CsA. Thus, TMC, whose mechanism of action is different from that of CsA or FK506, can be used as a potent T cell-specific immunosuppressor.     

Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A.

The immunosuppressive agents cyclosporin A (CsA) and FK 506 bind to distinct families of intracellular proteins (immunophilins) termed cyclophilins and FK 506-binding proteins (FKBPs). Recently, it has been shown that, in vitro, the complexes of CsA-cyclophilin and FK 506-FKBP-12 bind to and inhibit the activity of calcineurin, a calcium-dependent serine/threonine phosphatase. We have investigated the effects of drug treatment on phosphatase activity in T lymphocytes. Calcineurin is expressed in T cells, and its activity can be measured in cell lysates. Both CsA and FK 506 specifically inhibit cellular calcineurin at drug concentrations that inhibit interleukin 2 production in activated T cells. Rapamycin, which binds to FKBPs but exhibits different biological activities than FK 506, has no effect on calcineurin activity. Furthermore, excess concentrations of rapamycin prevent the effects of FK 506, apparently by displacing FK 506 from FKBPs. These results show that calcineurin is a target of drug-immunophilin complexes in vivo and establish a physiological role for calcineurin in T-cell activation.     

Interferon alfa and gamma inhibit proliferation and collagen synthesis of human ito cells in culture

During the course of ongoing liver fibrogenesis, Ito cells acquire myofibroblastic features, proliferate, and synthesize increased amounts of extracellular matrix components. Interferon (IFN) alfa and IFN gamma have been shown to elicit antiproliferative and/or antifibrogenic effects in various cell cultures of mesenchymal origin. The aim of this study was to investigate the effects of IFN-α and IFN-γ on cultured human myofibroblastic Ito cells (MFBIC) proliferation and collagen synthesis and secretion. Serum-stimulated incorporation of [³h]-thymidine into DNA of MFBIC was dose-dependently decreased by both cytokines. IFN-α (10⁴U/mL) and IFN-γ (10³U/mL) decreased DNA synthesis by 69% and 66%, respectively. Inhibition of cell proliferation was confirmed by cell counting. Similar results were observed when cell growth was stimulated with platelet-derived growth factor (PDGF-BB, PDGF-AA) or transforming growth factor (TGF)-βl. Collagen secretion per cell was inhibited by both cytokines, as assessed by [³h]hydroxyproline incorporation. After a 6-day treatment, IFN-γ showed a greater potency than IFN-α in inhibiting secretion of newly synthetized collagen (41% and 48% of control in the presence of 10² U/mL of IFN-γ and 10⁴U/ mL of IFN-α, respectively). Both IFN-α and IFN-γ concurrently decreased steady-state expression of type I and type III procollagen messenger RNAs (mRNAs) in quiescent MFBIC. Viability assays ruled out cytotoxic effects of the two molecules. Finally, both IFNs decreased smooth muscle α-actin (SMα-actin) expression, whether assayed by immunobloting or by Northern blot analysis. We conclude that IFN-α and IFN-γ inhibit proliferation as well as collagen synthesis in human MFBIC.     

他克莫司治疗移植肾慢性排斥的初步临床观察

目的：探讨他克莫司（FK506）、环孢素A（CsA)治疗移植肾慢性排斥（CR)的可行性及安全性。方法：40例同种异体肾移植患者肾功能减退经病理证实为CR，随机分为CsA切我为FK506组20例、继续使用CsA组20例。观察各组移植肾功能、肾小球滤过率、蛋白尿、血压、血脂变化及急性排斥（AR）发生率，治疗后随访12个月。结果：追踪12个月，FK506组16例移植肾功能稳定（80％）；3例行血液透析治疗，1例死亡，人存活率95％。CsA组15例移植肾功能稳定，3例行血液透析治疗，逆转成功率75％；2例死亡，人存活率90％。结论：FK506可以延缓慢性移植物失功。FK506的使用是安全和有效的。     

吡喹酮异构体对肝星状细胞系LX⁃2增殖及活化的作用

目的　观察左旋吡喹酮（L⁃PZQ）和右旋吡喹酮（D⁃PZQ）体外对人肝星状LX⁃2细胞系增殖及活化的作用差异。方法　利用转化生长因子⁃β（TGF⁃β）刺激激活LX⁃2细胞;采用CCK⁃8法检测以0 ~ 50 μg/mL梯度浓度吡喹酮刺激24 h后的LX⁃2细胞增殖情况;采用实时荧光定量PCR（qPCR）和免疫印迹试验检测15 mg/mL吡喹酮刺激LX⁃2细胞24 h 后的Ⅰ型胶原、Ⅲ型胶原和α⁃平滑肌肌动蛋白（α⁃SMA）基因表达水平和48 h后蛋白表达水平,以分析LX⁃2细胞活化情况。结果　L⁃PZQ和D⁃PZQ两药物各浓度组LX⁃2细胞存活率差异均有统计学意义（F = 6.119、79.180,P均< 0.05）;药物浓度< 30 μg/mL时,L⁃PZQ和D⁃PZQ对激活型LX⁃2增殖能力均无显著影响（P均> 0.05）;L⁃PZQ浓度> 50 μg/mL和D⁃PZQ浓度> 40 μg/mL时,均对激活型LX⁃2增殖能力产生抑制作用（P均< 0.05）;D⁃PZQ浓度为40 μg/mL和50 μg/mL时,对激活型LX⁃2增殖的抑制作用均强于L⁃PZQ（t = 3.419、8.776,P均< 0.05）。空白组、TGF⁃β诱导组、L⁃PZQ组和D⁃PZQ组LX⁃2细胞中Ⅰ型胶原、Ⅲ型胶原和α⁃SMA基因（F = 21.55、79.99、46.70,P 均< 0.05）和蛋白表达水平（F = 20.12、30.29、32.93,P 均< 0.05）差异均有统计学意义。L⁃PZQ对激活型LX⁃2细胞中Ⅲ型胶原和α⁃SMA基因表达水平具有显著抑制作用（P均< 0.05）,但对Ⅰ型胶原基因表达水平及Ⅰ型胶原、Ⅲ型胶原和α⁃SMA蛋白表达水平均无显著影响（P均> 0.05）;D⁃PZQ对激活型LX⁃2细胞中Ⅰ型胶原、Ⅲ型胶原和α⁃SMA基因和蛋白表达水平均有显著抑制作用（P均< 0.05）;D⁃PZQ对激活型LX⁃2细胞中Ⅰ型胶原、Ⅲ型胶原和α⁃SMA基因表达抑制作用强于L⁃PZQ（P均< 0.05）。结论　L⁃PZQ和D⁃PZQ对LX⁃2细胞增殖及活化均有一定抑制作用,且D⁃PZQ的抑制作用强于L⁃PZQ。     

Mycophenolate mofetil and FK506 have different effects on kidney allograft fibrosis in rats that underwent chronic allograft nephropathy

ABSTRACT: BACKGROUND: Tacrolimus (FK506) is associated with renal fibrosis in long-term use. Mycophenolatemofetil (MMF) can also inhibit or attenuate the progression of renal fibrosis. This study aimed to determine the different effects of FK506 and MMF on fibrosis-associated genes in the kidney in rats that underwent chronic allograft nephropathy (CAN). METHODS: Fisher (F344) kidneys were orthotopically transplanted into Lewis rat recipients. All recipients were given Cyclosporin A (CsA) 10 mg/kg-1.d-1 x 10 day and were then randomly divided into three oral treatment groups (n = 9 in each group): (1) the vehicle group was given vehicle orally; (2) the FK506 group was given 0.15 mg/kg-1.d-1 FK506; and (3) the MMF group was given 20 mg/kg-1.d-1 MMF. At 4, 8, and 12 weeks post-transplantation, serum creatinine (SCr), collagen deposition, Connective tissue growth factor (CTGF), alpha smooth muscle actin (alpha-SMA) and E-cadherin expressions were determined and hematoxylin-eosin (HE) and Periodic acid-Schiff (PAS) stains were performed. RESULTS: Renal function progressively deteriorated and showed typical CAN morphology in the vehicle and FK506 groups, while SCr and inflammatory infiltration (Banff score) showed a significant decrease in the MMF group after 8 weeks post-transplantation compared with those in the other groups (p < 0.05). Furthermore, expression levels of CTGF and alpha-SMA in the MMF group were significantly reduced, and the down-regulated expression of E-cadherin was abated (p < 0.05). CONCLUSIONS: MMF showed favorable effects on renal interstitial fibrosis, thus efficiently retarding the progression of CAN.     

胰岛素样生长因子-1对胃平滑肌细胞合成干细胞因子的作用

目的 探讨胰岛索样生长因子-1(IGF-1)对胃平滑肌细胞(SMC)及其合成的干细胞因子(SCF)的影响.方法 使用酶解法原代培养胃SMC,在进行-actin鉴定后,IGF-1组于细胞培养液中分别加入不同浓度的IGF-1(0、50、100、150ìg/L),抗IGF-1组于细胞培养液中加入IGF-1(100μg/L)和IGF-1α受体(IGF-1Rα)抗体(浓度分别为0、50、100,150μg/L),分别培养24 h.以四甲基偶氮唑盐(MTT)比色试验检测细胞增殖程度,以Western印迹法、逆转录(RT)-PCR、酶联免疫吸附试验(ELISA)检测细胞中及细胞上清中SCF的表达情况.结果 IGF-1可促进胃SMC增殖和SCF合成增加,离体实验中100μg/L可能是IGF-1促进胃SMC增殖和SCF合成的有效终浓度;IGF-1Rα抗体可抑制胃SMC增殖和SCF合成,抑制作用呈浓度依赖性.结论 IGF-1介导胃SMC合成SCF增加.     

Crystal structure of human calcineurin complexed with cyclosporin A and human cyclophilin

Calcineurin (Cn), a Ca(2+)/calmodulin-dependent Ser/Thr protein phosphatase, is an important participant in signaling pathways that activate T cells. It is the target of the immunosuppressive drugs cyclosporin A (CsA) and FK506. These drugs bind proteins known as cyclophilin (Cyp) and FK506-binding protein, respectively, and the drug-protein complexes in turn inhibit Cn. We report the crystal structure of a Cyp/CsA/Cn ternary complex, determined to a resolution of 3.1 A. Residues 3-9 of CsA, particularly N-methyl leucines 4 and 6, and Trp-121 of Cyp form a composite surface for interaction with Cn. The hydrophobic interface buries two hydrogen bonds. The structure accounts clearly for the effects of mutations in Cn on CsA-resistance and for the way modifications of CsA alter immunosuppressive activity.