Proteolytic maturation of transforming growth factor-α

Summary Transforming growth factor- (TGF-) is a mitogenic peptide hormone produced extracellularly, by tumor cells, and by virally and chemically transformed cells in culture. TGF- is almost certainly derived from its precursor protein (pro-TGF-) by limited endoproteolysis, but physiologically relevant processing enzyme(s) of the pro-TGF- protein and the cellular or subcellular compartment in which processing takes place are not known with certainty. We previously detailed [Cappelluti, E. and Harris, R.B., Biochemistry, 32 (1993) 551] the discovery, characterization and purification of novel, elastase-like enzymes (molecular weight 38 000) from oncogenically transformed rat liver epithelial cells or cultural Schwann cells transfected with SV40-large T antigen. The elastase-like enzyme appeared to be specifically induced in the transformed epithelial cells compared with the level of enzyme in the nontransformed parental cells. In the intervening time, other elastase-like serine proteinases have been implicated in processing pro-TGF- in other human carcinoma cell lines. We now report that the elastase-like enzymes, purified from transformed Schwann or liver epithelial cells, are inhibited in a time- and concentration-dependent fashion with three differently substituted monocyclic -lactam-based compounds originally developed as specific inhibitors of polymorphonuclear leukocyte elastase, thus further supporting the elastase-like character of the putative pro-TGF- processing enzymes. We also report the presence of the elastase-like enzyme in two different human malignant mammary cell lines, but even though MCF-7 cells receiving high doses of radiation in vitro show an increased level of expression of TGF-, the elastase-like enzyme does not appear to be induced in these cells following irradiation.     

Epidermal growth factor treatment switches δ-opioid receptor-stimulated extracellular signal-regulated kinases 1 and 2 signaling from an epidermal growth factor to an insulin-like growth factor-1 receptor-dependent mechanism

δ-Opioid receptor (DOR)-induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) is mediated by the transactivation of epidermal growth factor (EGF) receptors. Here we demonstrate that in stably DOR-expressing human embryonic kidney (HEK) 293 (HEK/DOR) cells, down-regulation of EGF receptors by long-term EGF (0.1 μg for 18 h) treatment, but not by small interfering RNA, results in functional desensitization of EGF (10 ng/ml)-stimulated ERK1/2 signaling. In EGF receptor-desensitized (HEK/DOR(-EGFR)) cells, however, [d-Ala2,d-Leu?]enkephalin (1 μM) and etorphine (0.1 μM) retained their ability to stimulate ERK1/2 activation. The newly acquired signal transduction mechanism is insensitive to the EGF receptor blockers 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) and N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (CL-387,785), does not involve DOR internalization and activation of the focal adhesion kinase pp125FAK, but requires matrix metalloproteinase-dependent release of soluble growth factors. A supernatant transfer assay in which conditioned growth media of opioid-treated HEK/DOR and HEK/DOR(-EGFR) "donor" cells are used to stimulate ERK1/2 activity in DOR-lacking HEK293 wild type and HEK293(-EGFR) "acceptor" cells revealed that long-term EGF treatment produces a switch in the receptor tyrosine kinase (RTK) system transactivated by opioids. Using microfluidic electrophoresis, chemical inhibitors, phosphorylation-specific antibodies, and EGF receptor-deficient Chinese hamster ovary-K1 cells, we identified the release of an insulin-like growth factor-1 (IGF-1)-like peptide and activation of IGF-1 receptors in HEK/DOR(-EGFR) cells after DOR activation. A similar switch from a neurotrophic tyrosine kinase receptor type 1 to an IGF-1 receptor-dependent ERK1/2 signaling was observed for chronically nerve growth factor-treated neuroblastoma × glioma (NG108-15) cells. These results indicate that transactivation of the dominant RTK system in a given cellular setting may represent a general feature of opioids to maintain mitogenic signaling.     

Effect of transforming growth factor-beta on activity of connective tissue growth factor gene promoter in mouse NIH ／ 3T3 fibroblasts

AIM: To investigate the regulatory mechanism of transforming growth factor-betaon activity of connective tissue growth factor promoter in mouse NIH/3T3 fibroblasts. METHODS: The regulation fragment of the 5' flanking region of the human CTGF gene was linked to pGL3-Basic vector, a firefly luciferase reporter construct without promoter. The recombinant plasmid pCTGF-luc was transiently transfected to NIH/3T3 fibroblasts. The activity of CTGF promoter after treatment of TGF-β1…     

Lack of effect of growth hormone replacement therapy on CYP1A2 and xanthine oxidase activities in growth hormone–deficient children

Background and objectives The recombinant human growth hormone (rhGH) is being increasingly used for a number of metabolic alterations. GH is the main regulator of several hepatic drug metabolizing enzymes in rodents. In addition, GH could play a major role in defining the interface between pharmacogenetics and development. However, little is known about the effect of GH on the activity of hepatic enzymes in children. The aim of this study was to determine the effect of rhGH replacement therapy for 4 weeks on CYP1A2 and xanthine oxidase (XO) activities in children. Methods We used caffeine as a probe drug to assess the enzyme activities at two points in time: before starting GH treatment (day 0) and after 4 weeks on rhGH therapy (day A). A total of 31 GH-deficient children (age range: 4.1–13.1 years, mean age: 9.88±2.89 years) participated. Urinary concentrations of caffeine and metabolites were determined by high-performance liquid chromatography (HPLC) to calculate the metabolite ratios: (AFMU+1X+1U)/17U for CYP1A2 and 1U/(1X+1U) for XO. Results Four weeks of GH substitution did not importantly alter the markers of the enzyme activities measured in this study. Median values and 95% confidence intervals (CI) at baseline were 5.17 (3.87–5.59) for the CYP1A2 ratio and 0.62 (0.56–0.65) for the XO ratio. These values, after treatment, were 4.57 (3.90–5.97) for the CYP1A2 marker and 0.62 (0.59–0.67) for the XO ratio. Data comparison between periods showed lack of statistically significant differences (P>0.05). The relative changes measured by the ratios of medians and 90% CI were 1.14 (0.90–1.31) and 0.99 (0.94–1.06) for CYP1A2 and XO, respectively. Conclusions The absence of significant changes in the markers of enzyme activities CYP1A2 and XO suggests that rhGH replacement therapy of GH-deficient children for 4 weeks could not noticeably modify the efficacy or toxicity of substrates of these metabolic enzymes.     

Dauricine inhibits insulin-like growth factor-I-induced hypoxia inducible factor 1α protein accumulation and vascular endothelial growth factor expression in human breast cancer cells

Aim:

To investigate the effects of dauricine (Dau) on insulin-like growth factor-I (IGF-I)-induced hypoxia inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in human breast cancer cells (MCF-7).

Methods:

Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of Dau, followed by incubation with IGF-I for 6 h. HIF-1α and VEGF protein expression levels were analyzed by Western blotting and ELISA, respectively. HIF-1α and VEGF mRNA levels were determined by real-time PCR. In vitro angiogenesis was observed via the human umbilical vein endothelial cell (HUVEC) tube formation assay. An in vitro invasion assay on HUVECs was performed.

Results:

Dau significantly inhibited IGF-I-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression. However, Dau remarkably suppressed VEGF expression at both protein and mRNA levels in response to IGF-I. Mechanistically, Dau suppressed IGF-I-induced HIF-1α and VEGF protein expression mainly by blocking the activation of PI-3K/AKT/mTOR signaling pathway. In addition, Dau reduced IGF-I-induced HIF-1α protein accumulation by inhibiting its synthesis as well as by promoting its degradation. Functionally, Dau inhibited angiogenesis in vitro. Moreover, Dau had a direct effect on IGF-I-induced invasion of HUVECs.

Conclusion:

Dau inhibits human breast cancer angiogenesis by suppressing HIF-1α protein accumulation and VEGF expression, which may provide a novel potential mechanism for the anticancer activities of Dau in human breast cancer.     

Transforming growth factor-β/Smad signalling in diabetic nephropathy

Diabetic nephropathy (DN) is a major diabetic complication that is mediated by transforming growth factor (TGF)-β1 via Smad-dependent and -independent signalling pathways. Under diabetic conditions, many profibrotic factors, such as advanced glycation end-products and angiotensin II, can also activate the Smad signalling pathway via the extracellular signal-regulated kinase/p38 mitogen-activated protein kinase-Smad signalling cross-talk pathway. Thus, Smads act as signal integrators and interact with other signalling pathways to mediate DN. In the context of renal fibrosis, Smad3 is pathogenic, but Smad2 is protective. Deletion of Smad3 inhibits, whereas disruption of Smad2 upregulates, connective tissue growth factor and vascular endothelial growth factor expression and promotes both epithelial-myofibroblast and endothelial-myofibroblast transition. Smad7 plays a protective role in DN because deletion of Smad7 enhances, whereas overexpression of Smad7 inhibits, Smad3-mediated renal fibrosis and nuclear factor-κB-driven renal inflammation. Transforming growth factor-β1 activates Smad3 to regulate microRNAs that mediate renal fibrosis. Of these, miR-21 and miR-192 are upregulated, whereas the miR-29 and miR-200 families are downregulated. Targeting downstream TGF-β/Smad signalling by overexpressing Smad7- or Smad3-dependent microRNA related to fibrosis may represent a novel and effective strategy for the treatment of DN.     

SomatoKine: is there a use in treating growth disorders?

Growth hormone (GH) has been used to treat GH deficiency since the late 1950s, and recombinant GH has been available since 1985. GH is also approved to treat non-GH-deficient short stature, such as that seen in Turner syndrome, chronic renal insufficiency, Prader-Willi syndrome, children who are small for gestational age, and idiopathic short stature. There has been interest in using recombinant insulin-like growth factor I (IGF-I) to treat short stature, either alone or in combination with its binding protein, IGF binding protein (IGFBP)-3 (SomatoKine). IGF-I increases growth velocity in children with IGF deficiency, either as a result of growth hormone insensitivity syndrome (GHIS) or IGF-I gene deletion. However, there have been adverse events, particularly hypoglycemia, reported with administration of unbound IGF-I. In addition, the serum half-life of unbound IGF-I is shorter when administered to patients with GHIS, who have low serum concentrations of its primary binding protein IGFBP-3 than when administered to healthy individuals or to patients with an IGF-I gene deletion (who have normal levels of IGFBP-3). SomatoKine was developed to prolong the half-life and to counteract acute adverse events (particularly hypoglycemia) associated with IGF-I administration. SomatoKine appears to have a longer half-life in patients with GHIS than unbound IGF-I and fewer adverse events (including hypoglycemia) have been reported when administered to patients with diabetes.     

Basic fibroblast growth factor protected forebrain against ischemia-reperfusion damage in rats

目的：研究碱性成纤维细胞生长因子（ｂＦＧＦ）对大鼠脑缺血再灌注损伤的影响．方法：电灼闭塞椎动脉并夹闭颈动脉，使大鼠前脑缺血２０分钟后，放开双侧颈动脉再灌．用高压液相色谱法测定纹状体中多巴胺（ＤＡ）类物质含量并用原子吸收分光光度法测定前脑中钠、钾含量．结果：再灌６小时后，纹状体中ＤＡ含量由对照的（９９±１６）μｇ·ｇ－１（蛋白）减少至（７０±２０）μｇ·ｇ－１蛋白；脑含水量由７７３４％±０１９％增加到７９６％±０６％；钠含量由（９３±０６）ｍｇ·ｇ－１（脑干重）增加到（１０５±０６）ｍｇ·ｇ－１（脑干重）．再灌开始后即刻静注ｂＦＧＦ４５μｇ·ｋｇ－１·ｈ－１共３ｈ可阻止ＤＡ的减少和水、钠含量的升高．组织学分析也表明ｂＦＧＦ可减轻神经元所受损伤．结论：ｂＦＧＦ可以保护神经细胞对抗脑缺血再灌损伤．     

Is inhibition of cancer angiogenesis and growth by paclitaxel schedule dependent?

It has been speculated that weekly paclitaxel enhances antiangiogenesis and, hence, results in a greater inhibition of cancer growth than the 3-week schedule. We compared the weekly and 3-week schedules of paclitaxel in inhibiting angiogenesis, tumor growth and bone marrow hematopoiesis in a lung cancer model. Vehicle or paclitaxel was administered i.p. to three groups of nude mice bearing a human lung cancer. The vehicle was given weekly for six doses or every 3 weeks for two doses (Group A). Paclitaxel was administered at 20 mg/kg/week for six doses (Group B) or 60 mg/kg/3 weeks for two doses (Group C). The tumor growth rate was reduced by 50% equally in both the paclitaxel-treated groups. Intratumoral microvasculature was reduced by 70% in each paclitaxel-treated group. However, white blood cell count was significantly reduced in Group C in comparison with that of Group A or B. We conclude that in this model, angiogenesis and tumor growth were inhibited to the same extent when paclitaxel was administered on a weekly or 3-week schedule. Inhibition of tumor growth by paclitaxel was associated with suppression of angiogenesis. Weekly administration of paclitaxel resulted in a lower degree of leukopenia than with the 3-week schedule, mimicking the clinical setting.     

Production of neutralizing monoclonal antibody against human vascular endothelial growth factor receptor Ⅱ

AIM: To prepare neutralizing monoclonal antibody (mAb) against extracellular immunoglobulin (Ig)-like domain Ⅲ of vascular endothelial growth factor receptor KDR and study its biological activity. METHODS: Soluble KDR Ig domain III (KDR-Ⅲ) fusion protein was expressed in E Coli and purified from the bacterial periplasmic extracts via an affinity chromatography. Monoclonal antibodies against KDR-Ⅲ were prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [^3H]-thymidine incorporation assay were also used to detect the activity of anti-KDR mAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on vascular endothelial growth factor-induced mitogenesis of human endothelial cells. RESULTS: A monoclonal antibody, Ycom1D3 (IgG1), was generated from a mouse immunized with the recombinant KDR-Ⅲ protein. YcomlD3 bound specifically to both the soluble KDR-Ⅲ and the cell-surface expressed KDR. Ycom1D3 effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated KDR activation in human endothelial cells. Furthermore, the antibody efficiently neutralized VEGF-induced mitogenesis of human endothelial cells. CONCLUSION: Our results suggest that the anti-KDR mAb, Ycom1D3, has potential applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.     

Insulin-like growth factor I: an attractive option for chronic heart failure?

Chronic congestive heart failure is a syndrome with a poor prognosis. Currently, the only therapy providing the possibility of long term survival is heart transplantation. Therefore, new therapeutic strategies continue to be investigated. One such new approach may be the application of recombinant human insulin-like growth factor (IGF)-1. IGF-1 has both acute and long term cardiovascular effects. Acute administration of IGF-1 resulted in a reduction in afterload and positive inotropic effects in patients with heart failure. In vitro and animal studies have demonstrated that IGF-1 can stimulate myofibril formation. In addition, IGF-1 administration has beneficial metabolic effects. The benefits of prolonged IGF-1 therapy have yet to be investigated.