Sassan Hafizi

Gas6 and protein S. Vitamin K-dependent ligands for the Axl receptor tyrosine kinase subfamily.

Gas6 and protein S are two homologous secreted proteins that depend on vitamin K for their execution of a range of biological functions. A discrete subset of these functions is mediated through their binding to and activation of the receptor tyrosine kinases Axl, Sky and Mer. Furthermore, a hallmark of the Gas6–Axl system is the unique ability of Gas6 and protein S to tether their non receptor‐binding regions to the negatively charged membranes of apoptotic cells. Numerous studies have shown the Gas6–Axl system to regulate cell survival, proliferation, migration, adhesion and phagocytosis. Consequently, altered activity/expression of its components has been detected in a variety of pathologies such as cancer and vascular, autoimmune and kidney disorders. Moreover, Axl overactivation can equally occur without ligand binding, which has implications for tumorigenesis. Further knowledge of this exquisite ligand–receptor system and the circumstances of its activation should provide the basis for development of novel therapies for the above diseases.

Analysis of Gas6 in Human Platelets and Plasma

Objective—Gas6 is a member of the vitamin K-dependent protein family. Gas6-deficient mice were found to be resistant to thrombosis because of defective platelet function. Mouse Gas6 was demonstrated to be present in platelets and found to be involved in platelet aggregation. The aim of this study was to investigate the presence of Gas6 in human platelets and plasma and determine its role in platelet function. Methods and Results—The presence of Gas6 in human platelets and plasma was analyzed using sensitive immunologic methods. Mass spectrometry and ELISA were used to identify and quantify Gas6 in plasma. Gas6 was demonstrated to be present in human plasma, at a concentration determined to be 13 to 23 ng/mL (0.16 to 0.28 nM). Furthermore, plasma Gas6 levels were found to be lower in patients administered with warfarin. However, Gas6 was undetectable in human platelets. Conclusions—This is the first report to identify and quantify Gas6 in human plasma. However, Gas6 protein was not detected in human platelets, suggesting that any potential platelet-specific function could be because of Gas6 from the circulation. These findings open up new directions regarding the role of Gas6 in normal and pathophysiological situations such as inflammation, autoimmune disease, thrombosis and arteriosclerosis.

Differential expression of Axl and Gas6 in renal cell carcinoma reflecting tumor advancement and survival

Purpose: Overexpression of the receptor tyrosine kinase Axl is implicated in several cancers. Therefore, we conducted this study to determine the expression of Axl and its ligand Gas6 in various renal cell carcinoma (RCC) types and in oncocytoma. Experimental Design: Real-time quantitative reverse transcription-PCR was used to quantify tumor mRNA levels for Axl and Gas6 in a cohort (n = 221) of RCC patients. Serum levels of soluble sAxl and Gas6 proteins were measured using specific ELISA assays (n = 282). The presence of Axl protein in tumor tissue was evaluated by immunohistochemistry (n = 294). Results were correlated to tumor-associated variables, clinical biochemical tests, and patient survival. Results: Tumor Axl mRNA levels correlated independently to survival when assessed against tumor stage and grade. In the study group, the median cancer-specific survival of all RCC patients during 307 months of follow-up was 55 months (confidence interval, ±40.4). The 25% of patients with lowest tumor Axl mRNA levels had significantly better survival than the rest (P = 0.0005), with 70% of the patients still alive at the end of follow-up. In contrast, in patients with medium-high Axl mRNA, only 25% were alive at the end of follow-up. Tumor Gas6 mRNA levels correlated to survival, tumor-associated variables, and disease severity as did serum levels of soluble sAxl and Gas6 protein. However, no correlation between Axl protein in tumor tissue and survival was found. Conclusions: Axl and Gas6 expression in RCC are associated with tumor advancement and patient survival. In particular, low tumor Axl mRNA levels independently correlated with improved survival.

Endothelin-1 stimulates proliferation of human coronary smooth muscle cells via the ETA receptor and is co-mitogenic with growth factors

We investigated the effects of endothelin-1 (ET-1) on growth of cultured human coronary artery smooth muscle cells (cSMC). ET-1 alone stimulated DNA synthesis in growth-arrested cSMC as measured by [3H]thymidine incorporation, with a maximum 63 +/- 23% increase above control by 10(-7) M (P < 0.05). ET-1 (10(-7) M) also stimulated increases in cyclin D1 protein levels after 24 h, and in absolute cell number after 4 days. Furthermore, ET-1 stimulated protein synthesis (maximum 73 +/- 32% increase in [3H]leucine incorporation by 10(-7) M (P < 0.05)), as well as triggering intracellular calcium transients in human cSMC, as visualised under fura-2 fluorescence microscopy. The selective ET(A) receptor antagonist BQ123 inhibited the increases in DNA synthesis, cell number, protein synthesis and intracellular calcium concentration in response to ET-1, whereas the ET(B) receptor antagonist BQ788 had no such effects. Furthermore, the ET(B) agonist sarafotoxin 6c had no effect on cSMC DNA synthesis. In addition, co-incubation of ET-1 with threshold concentrations of the growth factors, platelet-derived growth factor-BB (PDGF-BB), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), resulted in pronounced synergistic increases in DNA synthesis over that observed with the factors alone. In conclusion, we have shown that ET-1 stimulates proliferation of human cSMC via the ET(A) receptor and is also a co-mitogen with the growth factors tested. These findings indicate a role for ET-1 in the development of coronary intimal hyperplasia in man.

Interaction of Axl receptor tyrosine kinase with C1-TEN, a novel C1 domain-containing protein with homology to tensin.

Axl receptor tyrosine kinase is implicated in several malignancies and is the receptor for the vitamin K-dependent growth factor Gas6. From a yeast two-hybrid screen of protein-protein interactions with the Axl cytoplasmic domain, we detected a previously uncharacterised SH2 domain-containing protein. We cloned two novel splice variants of this protein that give rise to 1409- and 1419-amino acid proteins, differing only in their N-terminal residues and yielding a 150-kDa protein product by in vitro translation. The Axl-interacting C-terminus contains a tandem SH2 and PTB domain combination homologous to the focal adhesion protein tensin. We detected interaction of Axl with both domains in mammalian cells by co-immunoprecipitation and two-hybrid analyses. In addition, the protein possesses an N-terminal putative phorbol ester-binding C1 domain as well as a central tyrosine phosphatase motif. Thus, we have named the protein C1 domain-containing phosphatase and TENsin homologue (C1-TEN). Northern blot analysis of C1-TEN in human tissues revealed highest expression in heart, kidney, and liver. In summary, we have identified a novel multi-domain intracellular protein that interacts with Axl and which may furthermore be involved in other signal transduction pathways.

C1-TEN is a negative regulator of the Akt/PKB signal transduction pathway and inhibits cell survival, proliferation, and migration

We have previously identified C1 domain‐containing phosphatase and TENsin homologue (C1‐TEN) as being an intracellular binding partner for Axl receptor tyrosine kinase (RTK). C1‐TEN is a tensin‐related protein that houses an N‐terminal region with predicted structural similarity to PTEN. Here, we report our observations on the effects of ectopic expression of C1‐TEN in HEK293 cells, which resulted in profound molecular and phenotypic changes. Stable expression of C1‐TEN altered cellular morphology, with less cell spreading and weaker filamentous actin staining. Cells overexpressing C1‐TEN were inhibited greatly in their proliferation and migration rates as compared with mock‐transfected cells. Furthermore, serum starvation‐induced apoptosis caused a twofold increase in caspase 3 activity in C1‐TEN‐overexpressing cells vs. mock cells. In addition, C1‐TEN‐overexpressing cells showed a markedly reduced phosphorylation of Akt/PKB kinase and its substrate GSK3, as well as reduced Akt enzymatic activity. No such effects on JNK were observed. Also, serum‐stimulated activation of Akt was delayed in C1‐TEN‐overexpressing cells, while no difference in profile of ERK activation was observed. Furthermore, cells expressing a C1‐TEN mutant where the putative phosphatase active site cysteine at position 231 was substituted for a serine displayed full restoration of both cell proliferation and Akt activation. In conclusion, C1‐TEN appears to be a novel intracellular phosphatase that negatively regulates the Akt/PKB signaling cascade, and is similar to its relative PTEN in this respect. However, the particular domain organization of C1‐TEN may enable it to regulate RTK and other signaling complexes that are linked to Akt/PKB signaling in a unique manner.

Expression of functional angiotensin-converting enzyme and AT1 receptors in cultured human cardiac fibroblasts

BACKGROUND Angiotensin II (Ang II) has been implicated in the development of cardiac fibrosis. The aims of the present study were to examine expression and activity of ACE and of angiotensin receptors in human cardiac fibroblasts cultured from dilated cardiomyopathic and ischemic hearts. The effects of Ang II on fibroblasts were also investigated. METHODS AND RESULTS Human cardiac fibroblasts were cultured from ventricular and atrial myocardium and characterized immunohistochemically. Expression of ACE and the angiotensin AT1 receptor was demonstrated in cardiac fibroblasts by reverse transcriptase-polymerase chain reaction and radioligand binding. Functional ACE activity, measured by radiolabeled substrate conversion assay, was detected in both ventricular (Vmax. Km-1. mg-1, 0.031+/-0.010; n=13) and atrial (0. 034+/-0.012; n=6) fibroblasts. Fibroblast ACE activity was increased after 48 hours of treatment with basic fibroblast growth factor, dexamethasone, and phorbol ester. Ang II did not affect DNA synthesis but stimulated [3H]proline incorporation in cardiac fibroblasts (20.0+/-4.0% increase above control by 10 micromol/L; P<0.05, n=7), which was abolished by losartan 10 micromol/L but not PD123319 1 micromol/L. Ang II also stimulated a rise in intracellular calcium (basal, 56+/-1 nmol/L; Ang II, 355+/-24 nmol/L) via the AT1 receptor, as shown by complete inhibition with losartan. CONCLUSIONS We have demonstrated expression and activity of ACE and AT1 receptor in cultured human cardiac fibroblasts. In addition, cardiac fibroblasts respond to Ang II with AT1 receptor-mediated collagen synthesis. The presence of local ACE and AT1 receptors in human fibroblasts suggests their involvement in the development of cardiac fibrosis.

Profibrotic effects of endothelin-1 via the ETA receptor in cultured human cardiac fibroblasts.

Background/Aims: Endothelin-1 (ET-1) has been implicated in pathologic remodelling and tissue repair processes in the heart. We investigated the effects of ET-1 on growth and collagen synthesis responses in cardiac fibroblasts isolated from human hearts. We also studied the receptor subtype(s) mediating such responses and the factors regulating their expression. Methods: Fibroblasts were isolated from cardiac transplant recipient hearts and characterised by immunocytochemistry. Serum-starved cells were exposed to ET-1 and incorporation of [3H]proline and thymidine were measured as indexes of collagen and DNA synthesis respectively. Blocking experiments utilised the selective ETA receptor antagonist BQ123 and the ETB antagonist BQ788. Results: ET-1 elicited a potent collagen synthesis response in cardiac fibroblasts, with a maximum 29±5% increase that was abolished by BQ123. Cardiac fibroblasts responded to ET-1 with a concentration-dependent decrease in DNA synthesis rate. The effects of ET-1 were similar to those of TGF-β. Radioligand binding studies revealed the presence of high-affinity ET-1 binding sites on these cells, which were upregulated by treatment with the growth factors PDGF and EGF but downregulated by TGF-β. Conclusions: These results therefore implicate ET-1 as a trophic agent in the human heart with the ability to influence the development of cardiac fibrosis.

Induction of nitric oxide synthase in human vascular smooth muscle: interactions between proinflammatory cytokines

OBJECTIVE We have attempted to demonstrate the induction of inducible nitric oxide synthase in human vascular tissue and define the capacity of different cytokines to induce this enzyme. METHODS Segments of human arteries were stimulated with lipopolysaccharide (10 micrograms/ml), interleukin-1 beta (5 U/ml), tumor necrosis factor-alpha (10 U/ml), and interferon-gamma (200 U/ml). Cytokines were either used alone or in certain combinations, as well as in the presence of L-NG-monomethyl-arginine (100 mumol/l) or cycloheximide (1 mumol/l). Induction was assessed by measurement of mRNA expression, immunocytochemical localisation of the expressed protein, nitric oxide synthase activity and levels of nitrite, a product of nitric oxide formation. RESULTS PCR analysis showed the presence of mRNA for iNOS in stimulated samples which could be inhibited by cycloheximide. There was positive staining with an antibody against human iNOS in the media of stimulated vessel segments. Stimulated segments were also shown to contain Ca(2+)-independent nitric oxide synthase activity. The cytokines and lipopolysaccharide together gave a significant rise in levels of nitrite in the medium after 36 and 48 h, which was inhibited by L-NG-monomethyl-arginine and cycloheximide. Only interferon-gamma incubated alone was capable of increasing nitrite levels. This effect was enhanced by co-incubation with either interleukin-1 beta, tumor necrosis factor-alpha or lipopolysaccharide. CONCLUSION We have shown that increased production of nitrite by human vascular tissue in response to cytokines is associated with induction of iNOS as shown at the molecular and protein levels, and further supported by the presence of increased Ca(2+)-independent nitric oxide synthase activity following cytokine stimulation.

Tensin3 is a negative regulator of cell migration and all four Tensin family members are downregulated in human kidney cancer.

BACKGROUND The Tensin family of intracellular proteins (Tensin1, -2, -3 and -4) are thought to act as links between the extracellular matrix and the cytoskeleton, and thereby mediate signaling for cell shape and motility. Dysregulation of Tensin expression has previously been implicated in human cancer. Here, we have for the first time evaluated the significance of all four Tensins in a study of human renal cell carcinoma (RCC), as well as probed the biological function of Tensin3. PRINCIPAL FINDINGS Expression of Tensin2 and Tensin3 at mRNA and protein levels was largely absent in a panel of diverse human cancer cell lines. Quantitative RT-PCR analysis revealed mRNA expression of all four Tensin genes to be significantly downregulated in human kidney tumors (50-100% reduction versus normal kidney cortex; P<0.001). Furthermore, the mRNA expressions of Tensins mostly correlated positively with each other and negatively with tumor grade, but not tumor size. Immunohistochemical analysis revealed Tensin3 to be present in the cytoplasm of tubular epithelium in normal human kidney sections, whilst expression was weaker or absent in 41% of kidney tumors. A subset of tumor sections showed a preferential plasma membrane expression of Tensin3, which in clear cell RCC patients was correlated with longer survival. Stable expression of Tensin3 in HEK 293 cells markedly inhibited both cell migration and matrix invasion, a function independent of putative phosphatase activity in Tensin3. Conversely, siRNA knockdown of endogenous Tensin3 in human cancer cells significantly increased their migration. CONCLUSIONS Our findings indicate that the Tensins may represent a novel group of metastasis suppressors in the kidney, the loss of which leads to greater tumor cell motility and consequent metastasis. Moreover, tumorigenesis in the human kidney may be facilitated by a general downregulation of Tensins. Therefore, anti-metastatic therapies may benefit from restoring or preserving Tensin expression in primary tumors.