Product Of Growth Arrest-specific Gene Research Articles

Involvement of Gas6 protein in mouse model of lipopolysaccharide-induced lung inflammation

Background: Gas6 is a product of growth arrest-specific gene 6 and is mostly expressed by the growth-arrested organs and is implicated in thrombosis, various inflammation, and age-related diseases. However, its role in chronic obstructive pulmonary disease has not been investigated yet. Methods: We developed lipopolysaccharide-induced mouse model of chronic lung inflammation to study its involvement. Therefore, male C57BL/6 mice (20–25 g) were grouped into control and LPS group; each group having 15 animals each. LPS group was nebulized with LPS (1 mg/ml) for 45 min, once a week for 8 weeks to induce chronic inflammatory conditions in the lungs by use of nebulizer. The mice model was validated by assessing infiltration of various immune cells (T-cells, B-cells, and neutrophils) into the lungs and pro-inflammatory and anti-inflammatory cytokine profile by cytokine bead array kit by flow cytometry. Lung deterioration was assessed by lung histology. The level of gas6 protein from the lung homogenate was measured by quantikine enzyme-linked immunosorbent assay kit. Furthermore, lung homogenate was analyzed by Western blotting for expression profile of gas6 protein. Results: There was significant increase in the level of T-cells (19±8% vs 65±11%), B-cells (24±7% vs 64±10%), and neutrophils (22±9% vs 57±10%) and significant increase in the level of tumor necrosis factor (200 ± 17 pg/ml vs. 1222 ± 152 pg/ml) and IL-6 (106 ± 13 pg/ml vs. 448 ± 122 pg/ml). Lung deterioration was observed in LPS group. We observed significant increase in the level of gas6 protein in lung homogenate in LPS group (0.16 ± 0.1 ng/ml vs. 4.2 ± 0.1 ng/ml). Furthermore, we found significant increase in gas6 protein in the lung homogenate of LPS-treated group by Western blotting. Conclusion: The current study establishes the involvement gas6 protein in lung inflammation.

Testicular immunomodulation and orchitis: Roles of Tyro3/ Axl/Mer receptor tyrosine kinases and pattern recognition receptors

Human infertility affects approximately 15% of couples at reproductive age worldwide. Male factors contribute up to 45% of infertility cases. Inflammatory conditions in the male genital system are responsible for about 10% of male infertility in developed countries, and the incidence of immunological infertility can be considerably higher in developing countries where medical cares and environment conditions are poor. In particular, immunological disorders in the testis freguently cause infertility because it is a organ where spermatogenesis carries out. The testis possesses a special immune environment because of its immunoprivileged status and innate immune system. Testicular immune privilege is essential for the protection of male germ cells from detrimental immune responses, whereas the local innate immune system plays a crucial role in the testicular defense against microbial infections. Most of testicular cells are involved in the regulation of testicular immune homeostasis. Disruption of testicular immune homeostasis may result in orchitis, one aetiological factor of male infertility. Multiple mechanisms cooperatively regulate testicular immune homeostasis. Recent studies have revealed important roles of Tyro3/Axl/Mer (TAM) receptor tyrosine kinases and pattern recognition receptors (PRRs) in regulating testicular immune environment. TAM receptors belong to one subfamily of receptor tyrosine kinases. The product of growth arrest-specific gene 6 (Gas6) is common ligand of TAM receptors. TAM and Gas6 are expressed in the testis. TAM triple knockout mice are male sterile and develop autoimmune orchitis. TAM receptors regulate testicular immune homeostasis via three mechanisms: (i) PRR-initiated innate immune responses in Sertoli and Leydig cells are inhibited by TAM/Gas6 signaling, which reduce inflammatory cytokine production in the testis; (ii) TAM receptors promote phagocytosis of apoptotic germ cells by Sertoli cells. The phagocytic clearance of apoptotic germ cells by Sertoli cells can remove germ cell antigens, thereby preventing endogenous immune response; (iii) TAM receptors favor systemic immune tolerance to male germ cells. Experimental autoimmune orchitis (EAO) is a testis-specific autoimmune inflammation in rodent animals after immunization with male germ cell antigens. Axl and Mer double knockout mice are susceptible to EAO induction, suggesting Axl and Mer cooperatively regulate the systemic immune tolerance to male germ cell antigens. Although the testis is an immunoprivileged organ, it can be infected by a broad spectrum of microbial pathogens. However, the testis usually recovers from microbial infections without evident inflammatory response, suggesting that the testis adopts an effective local innate defense system against invading microorganisms. Recognition of microbial pathogens by PRRs initiates innate immune responses, which is the first line of the host defense against invading microbes. Several subfamilies of PRRs are expressed in the testis. In particularly, major testicular cells, including Sertoli, Leydig, and male germ cells, express various PRRs. PRRs can be activated in testicular cells by pathogen molecular patterns, thereby initiating innate immune responses. These observations indicate that testicular cells are equipped with innate immune machinery and may be involved in the testicular defense against microbial infections. This article describes role of TAM receptors and PRRs in regulating immune environment in the testis, and speculates aspects of which need priorizing in future investigation.

Assay of serum growth arrest-specific gene product 6 and stromal cell-derived factor isoform-1 in patients with diabetic retinopathy

Background Diabetic retinopathy (DR) is a progressive vision-threatening complication of diabetes mellitus (DM),but its pathogenic mechanism is still unclear.Researches showed that growth arrest-specific gene product 6 (Gas6) /TAM system participates in pathogenesis and development of DR,and stromal-derived factors (SDF) vary in 2-type DM patients.However,whether Gas6/TAM and SDF-1 are associated with DR is below understood.Objective This study was to determine the relationship between the staging of DR and the levels of serum Gas6,SDF-1α and SDF-1β in DM patients.Methods A prospective cohord study was designed in this study.Ninety 2-type DM patients were included in the 3rd Affiliated Hospital of Southern Medical University Hospital from January to August in 2013.The patients were grouped into the non-diabetic retinopathy (NDR) group,background DR group (BDR) and proliferative DR (PDR) group,with 30 for each group.Thirty normal volunteers were enrolled in the same hospital and same period.The periphery blood 2 ml was collected from all the subjects under the consent inform.The levels of serum Gas6,SDF-1α and SDF-1β were assayed by ELISA,and leukocytes,neutrophils,plasma triglycerides (TG),total cholesterol (CHOL),high density lipoprotein cholesterol (H DL-C) and low density lipoprotein cholesterol (LDL-C) levels were detected and compared among the 4 groups.The correlations of serum Gas6,SDF-1 α and SDF-1 β changes with blood inflammatory cells and blood lipid were analyzed.Results The plasma CHOL concentrations were 4.93(4.14,5.44),5.02(4.35,5.69),4.54(3.85,5.93) and 5.99(5.11,6.89)mmol/L in the normal control group,NDR group,BDR group and PDR group,respectively,and the blood CHOL concentrations were significantly higher in the PDR group than those of the normal control group,NDR group,BDR group (P =0.002,P =0.007,P =0.006).White blood cell counts in the normal control group,BDR group were higher than those of the PDR group (P =0.034,P =0.015),neutrophil counts in the BDR group were higher than those of the PDR group (P =0.024),HDL-C in the NDR group was higher than that in the PDR group (P =0.032).LDL-C in the PDR group was higher than that in the normal control group.Compared with the normal control group,serum Gas6 levels were significantly lower in the NDR group and BDR group (P =0.048,P =0.006),and the serum Gas6 level showed an insignificant increase in the PDR group in comparison with BDR group (P =0.297).Serum SDF-1α levels in the PDR group was significantly higher than that in the BDR group (P =0.033) ;serum SDF-1β levels in the PDR group,BDR group were significantly higher than that in the NDR group (P =0.011,P =0.008) and normal control group (P =0.030,P =0.002).Weaker positive correlation was observed between the serum Gas6 and CHOL,TG,LDL-C levels (r=0.285,r=0.200,r=0.241,all at P＜0.05),between SDF-1α and SDF-1β (r=0.190,P＜0.05) as well as between SDF-1β and white blood cell (r=0.183,P＜0.05).Serum Gas6 served as dependent variable,while white blood cell,neutrophil,CHOL,TG,HDL-C,LDL-C,SDF-1α,SDF-1β served as independent variables,multiple stepwise regression analysis showed Gas6 =170.791 + 5.283CHOL (F =5.021,P =0.027).Conclusions Serum Gas6,SDF-1α and SDF-1β probably participate in the development of DR in 2-type diabetic patients.Gas6,SDF-1 α,SDF-1 β may play roles by affecting blood glucose level,angiogenesis,inflammatory cells and blood lipid metabolism. Key words: Growth arrest-specific gene 6; Stromal cell-derived factor-1; Diabetes mellitus; Complication; Diabetic retinopathy

Up-Regulation of Soluble Axl and Mer Receptor Tyrosine Kinases Negatively Correlates with Gas6 in Established Multiple Sclerosis Lesions

Multiple sclerosis is a disease that is characterized by inflammation, demyelination, and axonal damage; it ultimately forms gliotic scars and lesions that severely compromise the function of the central nervous system. Evidence has shown previously that altered growth factor receptor signaling contributes to lesion formation, impedes recovery, and plays a role in disease progression. Growth arrest-specific protein 6 (Gas6), the ligand for the TAM receptor tyrosine kinase family, consisting of Tyro3, Axl, and Mer, is important for cell growth, survival, and clearance of debris. In this study, we show that levels of membrane-bound Mer (205 kd), soluble Mer ( approximately 150 kd), and soluble Axl (80 kd) were all significantly elevated in homogenates from established multiple sclerosis lesions comprised of both chronic active and chronic silent lesions. Whereas in normal tissue Gas6 positively correlated with soluble Axl and Mer, there was a negative correlation between Gas6 and soluble Axl and Mer in established multiple sclerosis lesions. In addition, increased levels of soluble Axl and Mer were associated with increased levels of mature ADAM17, mature ADAM10, and Furin, proteins that are associated with Axl and Mer solubilization. Soluble Axl and Mer are both known to act as decoy receptors and block Gas6 binding to membrane-bound receptors. These data suggest that in multiple sclerosis lesions, dysregulation of protective Gas6 receptor signaling may prolong lesion activity.

GAS6-induced signaling in human endothelial cells is mediated by FOXO1a.

Growth Arrest Specific gene product 6 (gas6) is a gamma-carboxylated protein that protects endothelial cells against apoptosis. Gas6 has previously been shown to induce phospatidyl-3-inositol-kinase (PI3K)/Akt signaling. Other studies have demonstrated a link between PI3K/Akt signaling and forkhead transcription factors in endothelial cells. To test the hypothesis that gas6 promotes cell survival via a forkhead-dependent pathway. Treatment of serum-starved human umbilical vein endothelial cells (HUVECs) with gas6 induced time-dependent phosphorylation and nuclear exclusion of FOXO1a. This effect was suppressed by the PI3K inhibitor wortmannin, demonstrating that FOXO1a phosphorylation is PI3-kinase dependent. Transduction of HUVECs with a phosphorylation-resistant form of FOXO1a [triple mutant (TM)-FOXO1a] abrogated the pro-survival effect of gas6 on serum-starved endothelial cells. Finally, treatment of serum-starved HUVECs with gas6 resulted in a reduction of FOXO1a transcriptional activity and downregulation of the pro-apoptotic gene, p27(kip1). Taken together, these findings suggest that gas6 protects endothelial cells from apoptosis by a mechanism that involves PI3K-Akt-dependent inactivation of FOXO1a.

Role of Gas6 in erythropoiesis and anemia in mice

Many patients with anemia fail to respond to treatment with erythropoietin (Epo), a commonly used hormone that stimulates erythroid progenitor production and maturation by human BM or by murine spleen. The protein product of growth arrest-specific gene 6 (Gas6) is important for cell survival across several cell types, but its precise physiological role remains largely enigmatic. Here, we report that murine erythroblasts released Gas6 in response to Epo and that Gas6 enhanced Epo receptor signaling by activating the serine-threonine kinase Akt in these cells. In the absence of Gas6, erythroid progenitors and erythroblasts were hyporesponsive to the survival activity of Epo and failed to restore hematocrit levels in response to anemia. In addition, Gas6 may influence erythropoiesis via paracrine erythroblast-independent mechanisms involving macrophages. When mice with acute anemia were treated with Gas6, the protein normalized hematocrit levels without causing undesired erythrocytosis. In a transgenic mouse model of chronic anemia caused by insufficient Epo production, Gas6 synergized with Epo in restoring hematocrit levels. These findings may have implications for the treatment of patients with anemia who fail to adequately respond to Epo.

Growth arrest-specific protein 6 plasma concentrations during septic shock

IntroductionThe product of growth arrest-specific gene 6 (Gas6) is a vitamin K dependent protein that is secreted by leucocytes and endothelial cells in response to injury and participates in cell survival, proliferation, migration and adhesion. Our purpose was to investigate plasma Gas6 concentration and its relation to organ dysfunction in patients with septic shock.MethodsForty-five patients with septic shock admitted to a medical adult intensive care unit were enrolled. Plasma Gas6 concentration was determined using enzyme-linked immunosorbent assay at days 1, 3, 7 and 14.ResultsThe median (interquartile range) Gas6 concentration was 51 (5 to 95) pg/ml at admission. A positive correlation (Spearman rank-order coefficient [rs] = 0.37, P = 0.01) was found between Gas6 level and Sepsis-related Organ Failure Assessment score. Patients requiring renal support had higher Gas6 concentration that those without need for haemofiltration (76.5 [52 to 164] pg/ml versus 10.5 [1.5 to 80.5] pg/ml; P = 0.04). Moreover, there was a positive correlation between Gas6 and aspartate transaminase (rs = 0.42, P = 0.006) and between Gas6 and prothrombin time (rs = 0.45, P = 0.02). Although there was a progressive decline in Gas6 concentration in survivors (analysis of variance, P = 0.01), nonsurvivors exhibited persistently elevated Gas6. However, the two populations diverged only after day 7 (P = 0.04).ConclusionPlasma concentrations of Gas6 correlate with disease severity, especially with renal and hepatic dysfunction, in septic shock.

GAS-6 Rescues Endothelial Cells from Apoptosis through FOXO1 Inactivation.

Growth Arrest Specific gene product 6 (GAS-6), a γ-carboxylated protein expressed in quiescent fibroblasts and endothelial cells, exerts an anti-apoptotic function by binding to the receptor tyrosine kinase Axl. Recently, our laboratory has demonstrated that gas6-Axl interactions activate PI3-kinase with subsequent Akt phosphorylation during gas6-mediated protection from apoptosis. The current study explores further the mechanism by which this survival mechanism is achieved. FOXO1 is a member of the Forkhead family of transcription factors that plays a role in the expression of pro-apoptototic genes. Phosphorylation of FOXO1 at Thr24, Ser256 and Ser319 results in phospho-FOXO1 translocation from the nucleus to the cytoplasm, with consequent suppression of FOXO1 transcriptional activity and inhibition of apoptosis. In the present study we show, for the first time, that the treatment of serum-starved endothelial cells with 100 ng/ml of GAS-6 induces FOXO1 phosphorylation in a time dependent manner. Phosphorylated FOXO1 is translocated from the nucleus to the cytoplasm as evidenced by Western blot analysis of both nuclear and cytoplasmic extracts. Using fluorescence microscopy, FOXO1 is found predominantly in the nucleus during apoptosis induced by serum starvation. Upon gas6 stimulation, phosphorylated FOXO1 is translocated to the cytoplasm (see Figure 1). It is suggested that anti-apoptotic genes are then released from suppression and are thereby able to mediate cell survival. Both FOXO1 phosphorylation and translocation are suppressed by Wortmannin, a PI3-kinase inhibitor demonstrating that FOXO1 phosphorylation is PI3-kinase dependent. These results provide mechanistic insight of how gas6 rescues endothelial cells from serum-starvation-induced apoptosis. [Display omitted]

Gas6, warfarin, and kidney diseases

The product of growth arrest-specific gene 6 (Gas6) is a unique vitamin K-dependent autocrine growth factor for mesangial cells, and warfarin inhibits mesangial cell proliferation by interfering with the activation process of Gas6. A recent series of studies has revealed the in vivo roles of Gas6 and its receptor Axl in the progression of acute and chronic glomerulonephritis, diabetic nephropathy, chronic allograft rejection, and human kidney diseases. This review summarizes these studies and discusses the possible interventions targeting the Gas6/Axl pathway to prevent the progression of kidney diseases.

The role of the vitamin K-dependent growth factor Gas6 in glomerular pathophysiology.

The product of growth arrest-specific gene 6 (gas6) is a unique vitamin K-dependent growth-potentiating factor for vascular smooth muscle cells, and anticoagulant warfarin inhibits the activation process of the protein. It has been reported that Gas6 is also a mitogen for mesangial cells, and that warfarin inhibits mesangial cell proliferation by blocking the activation of Gas6. A recent series of studies has revealed the in-vivo roles of Gas6 and its receptor Axl in the progression of various kidney diseases. This review summarizes these studies and discusses the possible interventions targeting the Gas6/Axl pathway to prevent the progression of kidney diseases. The expression of Gas6 and Axl is upregulated in an acute model of glomerulonephritis in rats, and the interference of the Gas6/Axl pathway by warfarin or the extracellular domain of Axl inhibits the progression of diseases. Induction of chronic glomerulonephritis in Gas6 mice results in less mortality, proteinuria, and histological changes of kidneys compared to wild-type mice. Administration of recombinant Gas6 reverses these phenotypes. Expression of Gas6 is also upregulated in streptozotocin-induced diabetic nephropathy, and administration of low-dose warfarin decreases albuminuria and hypertrophy of glomeruli. Possible roles of Gas6 are also reported in renal allograft dysfunction of rats and humans. The importance of the Gas6/Axl pathway has been implicated in many types of kidney disease. Further investigations on the role of the Gas6/Axl pathway in human kidney diseases and the development of specific antagonists targeting the pathway are warranted.

Ligand Recognition and Homophilic Interactions in Tyro3: STRUCTURAL INSIGHTS INTO THE Axl/Tyro3 RECEPTOR TYROSINE KINASE FAMILY

The receptor Tyro3 together with Axl and Mer form the Axl/Tyro3 family of receptor tyrosine kinases. Members of this family play essential roles in spermatogenesis, immunoregulation, and phagocytosis. Gas6, the product of growth arrest-specific gene, activates the kinase activity of all three receptors. Here, we report the first biochemical and structural characterization of a member of this family, namely of a fragment spanning the two N-terminal Ig domains of the extracellular part of human Tyro3. Its ligand binding specificity profile is identical to the activation profile of the native receptor. The 1.95-A crystal structure suggests a common ligand-binding site in this receptor family located at the interface of the Ig domains and unusually rich in cis-prolines. Furthermore, both in the crystal and in solution we observed the ligand-independent dimerization of the receptor fragment. This homophilic interaction emphasizes previous functional reports, which hinted that in addition to signal transduction, members of this family of receptors might participate in cell adhesion.

A subunit of the dynein regulatory complex in Chlamydomonas is a homologue of a growth arrest-specific gene product.

The dynein regulatory complex (DRC) is an important intermediate in the pathway that regulates flagellar motility. To identify subunits of the DRC, we characterized a Chlamydomonas motility mutant obtained by insertional mutagenesis. The pf2-4 mutant displays an altered waveform that results in slow swimming cells. EM analysis reveals defects in DRC structure that can be rescued by reintroduction of the wild-type PF2 gene. Immunolocalization studies show that the PF2 protein is distributed along the length of the axoneme, where it is part of a discrete complex of polypeptides. PF2 is a coiled-coil protein that shares significant homology with a mammalian growth arrest–specific gene product (Gas11/Gas8) and a trypanosome protein known as trypanin. PF2 and its homologues appear to be universal components of motile axonemes that are required for DRC assembly and the regulation of flagellar motility. The expression of Gas8/Gas11 transcripts in a wide range of tissues may also indicate a potential role for PF2-related proteins in other microtubule-based structures.

Crystal Structure of a C-terminal Fragment of Growth Arrest-specific Protein Gas6: RECEPTOR TYROSINE KINASE ACTIVATION BY LAMININ G-LIKE DOMAINS

Receptor tyrosine kinases of the Axl family are activated by Gas6, the product of growth arrest-specific gene 6. Gas6-Axl signaling is implicated in cell survival, adhesion, and migration. The receptor-binding site of Gas6 is located within a C-terminal pair of laminin G-like (LG) domains that do not resemble any other receptor tyrosine kinase ligand. We report the crystal structure at 2.2-A resolution of a Gas6 fragment spanning both LG domains (Gas6-LG). The structure reveals a V-shaped arrangement of LG domains strengthened by an interdomain calcium-binding site. LG2 of Gas6-LG contains two unusual features: an alpha-helix cradled by one edge of the LG beta-sandwich and a conspicuous patch of surface-exposed hydrophobic residues. Mutagenesis of some residues in this patch reduces Gas6-LG binding to the extracellular domain of Axl as well as Axl activation in glioblastoma cells, identifying a component of the receptor-binding site of Gas6.

Isolation and Properties of Gas8, a Growth Arrest-specific Gene Regulated during Male Gametogenesis to Produce a Protein Associated with the Sperm Motility Apparatus

Growth arrest-specific (Gas) genes are expressed during serum starvation or contact inhibition of cells grown in culture. Here we report the isolation and characterization of Gas8, a novel gene identified on the basis of its growth arrest-specific expression in murine fibroblasts. We show that production of Gas8 mRNA and protein occurs in adult mice predominantly in the testes, where expression is regulated during postmeiotic development of male gametocytes. Whereas a low level of Gas8 mRNA was detected by Northern blotting in testes of murine male neonates and young adolescents, Gas8 mRNA increased rapidly postmeiotically. In adult males, both Gas8 mRNA and protein reached steady state levels in testes that were 10-fold higher than in other tissues. Immunohistochemical analyses showed that Gas8 protein accumulates in gametocytes as they approach the lumen of seminiferous tubules and is localized to the cytoplasm of round spermatids, the tails of elongating spermatids, and mature spermatid tail bundles protruding into the lumen; in epididymal spermatozoa Gas8 protein was present in the flagella. However, premeiotic murine gametocytes lacked detectable Gas8 protein, as did seminiferous tubules in biopsy specimens from seven human males having cytological evidence of non-obstructive azoospermia secondary to Sertoli cell-only syndrome. Our findings, which associate Gas8 production developmentally with the later stages of spermatogenesis and spatially with the sperm motility apparatus, collectively suggest that this growth arrest-specific gene product may have a role in sperm motility. This postulated role for Gas8 is supported by our observation that highly localized production of Gas8 protein occurs also in the cilia of epithelial cells lining pulmonary bronchi and fallopian tubes and by the flagellar association of a Trypanosoma brucei ortholog of Gas8.

Genetic studies on the role of proteinases and growth factors in atherosclerosis and aneurysm formation.

Expression analysis and epidemiologic studies have provided indirect evidence that proteinases and growth factors play a role in the development of atherosclerosis and complications such as aneurysm formation and plaque rupture. Studies using genetically altered mice have proven to be an elegant tool to study the causal involvement of these factors in atherogenesis and to gain insight into the underlying mechanisms. Recently, proteinases of the plasminogen and matrix metalloproteinase (MMP) systems as well as their inhibitors have received much attention, and these studies together have emphasized the complexity of their role in vascular disease. This overview summarizes the current knowledge on plasminogen activator inhibitor-1 (PAI-1) in the progression of atherosclerosis and the influence of MMPs in aneurysm formation. In addition, a possible role for Gas6, the product of growth arrest-specific gene 6, in atherosclerotic lesion development is put into perspective.

Gas6 Regulates Mesangial Cell Proliferation through Axl in Experimental Glomerulonephritis

Proliferation of mesangial cells is a hallmark of glomerular disease, and understanding its regulatory mechanism is clinically important. Previously, we demonstrated that the product of growth arrest-specific gene 6 (Gas6) stimulates mesangial cell proliferation through binding to its cell-surface receptor Axl in vitro. We also showed that warfarin and the extracellular domain of Axl conjugated with Fc portion of human IgG1 (Axl-Fc) inhibit mesangial cell proliferation by interfering the Gas6/Axl pathway in vitro. In the present study, therefore, we examined in vivo roles of Gas6 and Axl in an experimental model of mesangial proliferative glomerulonephritis induced by the injection of anti-Thy1.1 antibody (Thy1 GN). In Thy1 GN, expression of Gas6 and Axl was markedly increased in glomeruli, and paralleled the progression of mesangial cell proliferation. Administration of warfarin or daily injection of Axl-Fc inhibited mesangial cell proliferation, and abolished the induction of platelet-derived growth factor-B mRNA and protein in Thy1 GN. Moreover, the anti-proliferative effect of warfarin was achieved at lower concentrations than those in routine clinical use. These findings indicate that the Gas6/Axl pathway plays a key role in mesangial cell proliferation in vivo, and could be a potentially important therapeutic target for the treatment of renal disease.

Promotion of the uptake of PS liposomes and apoptotic cells by a product of growth arrest-specific gene, gas6.

Gas6, a ligand of receptor tyrosine kinases Axl, Sky, and Mer, potentiates cell proliferation and prevents cell death. It also contains g-carboxylglutamic acid residues that mediate the interaction of some blood coagulation factors with negatively charged phospholipids. In our previous study, we demonstrated that Gas6 specifically binds to phosphatidylserine (PS) and links Axl-expressing cells to the PS-coated surface. In this study, to further understand the biological role of the interaction of Gas6 with PS, we examined the effect of Gas6 on the uptake of PS liposomes by macrophages. In vitro phagocytosis studies showed that Gas6 enhanced the uptake of PS liposomes approximately threefold and that the interaction of Gas6 with the surface of macrophages was essential for this enhancement. Analyses of the mechanism of the uptake of PS liposome suggested that Gas6 interacts with PS liposome via its N-terminal Gla domain and with macrophages via its C-terminal domain. Like that of PS liposomes, the uptake of apoptotic cells by macrophages was also enhanced, approximately twofold, in the presence of Gas6. These findings suggest that Gas6 may help phagocytic cells recognize cells with PS exposed on their surfaces, which is considered to be one of the mechanisms for clearing away dying cells. Thus, Gas6 may play a critical role in homeostasis by facilitating the clearance of PS-expressing cells.

Release of a product of growth arrest-specific gene 6 from rat platelets

A product of growth arrest-specific gene 6 (Gas6) is known to be synthesized by growth-arrested cells. In this study, we found that several rat tissues including platelets contain Gas6 and activation of the platelets with thrombin provoked the release of Gas6. ADP and collagen, which as well as thrombin stimulated release of ATP from platelets, also enhanced the release of Gas6, suggesting that the mechanism of its release was similar to that of ATP release. This study provides the first evidence of growth arrest-independent secretion of Gas6 and suggests the involvement of Gas6 in vascular diseases as well as hemostasis.

A product of growth arrest-specific gene 6 modulates scavenger receptor expression in human vascular smooth muscle cells

Although Gas6 is identified as a growth factor for vascular smooth muscle cells (VSMCs), its roles in these cells have not been clearly elucidated. To examine the role of Gas6 in atherosclerosis, we examined the effects of Gas6 on scavenger receptor family expression in VSMCs. Scavenger receptor class A, one of the scavenger receptor family members, was upregulated in VSMCs by Gas6. Furthermore, the atherogenic lipoprotein, oxidized LDL, induced Gas6 production in these cells. These results indicate that Gas6 plays an important role in foam cell formation in human VSMCs.

Expression profile of receptor-type protein tyrosine kinase genes in the human thyroid.

Protein tyrosine kinases (PTKs) play a role in regulating the growth and differentiated functions of thyroid cells and are probably involved in tumorigenesis of papillary-type thyroid carcinoma. To better understand the roles of PTKs in the physiology and pathophysiology of the thyroid, we analyzed the expression profile of receptor-type PTKs in normal human thyroid tissues. Highly conserved regions in the catalytic domains of receptor-type PTKs were amplified by RT-PCR using degenerate oligonucleotide primers. Nucleotide sequencing of about 100 clones identified 21 PTKs, including 16 receptor type and 5 nonreceptor type; no novel PTK was identified. Insulin-like growth factor I receptor, platelet-derived growth factor receptor (PDGFR), TrkE, Axl, epidermal growth factor receptor, etc., appear to be the most abundant receptor-type PTKs in the thyroid; many of which (PDGFR, TrkE, Axl, etc.) have never previously been demonstrated to be expressed in the thyroid. The expression of messenger RNAs (mRNAs) for PDGFR, axl, and trkE in normal thyroid cells was confirmed by Northern blot analysis, and interestingly, the expression levels of PDGFR and trkE mRNAs were decreased in all three thyroid carcinoma cell lines examined (FRO, WRO, and NPA), whereas axl mRNA and protein were overexpressed in 2 of 3 thyroid carcinoma cell lines (FRO and WRO) compared with that in normal tissue. The axl gene was, however, neither amplified nor rearranged. The biological activity of the ligand for Axl, the product of growth arrest-specific gene 6 (Gas6), was then evaluated, demonstrating modest mitogenic activity in thyroid carcinoma cells overexpressing Axl. Furthermore, gas6 mRNA was expressed in FRO cells. Thus, we here identify a variety of PTKs expressed in the thyroid gland, many of which may participate in the regulation of thyroid cell function. Variable expression levels of some PTKs in normal and cancerous cells suggest that there may be an imbalance and disarray of phosphorylation events in thyroid carcinoma cells. Furthermore, Gas6 is identified as a novel growth factor for thyroid carcinoma cells overexpressing Axl receptor tyrosine kinase.