Preventive and chronic mineralocorticoid receptor antagonism is highly beneficial in obese SHHF rats.

BACKGROUND AND PURPOSE: Mineralocorticoid receptor (MR) activation contributes to heart failure (HF) progression. Its overactivity in obesity is thought to accelerate cardiac remodelling and HF development. Given that MR antagonists (MRA) are beneficial in chronic HF patients, we hypothesized that early MRA treatment may target obesity-related disorders and consequently delay the development of HF. EXPERIMENTAL APPROACH: Twenty spontaneously hypertensive HF dyslipidaemic obese SHHF(cp/cp) rats and 18 non-dyslipidaemic lean SHHF(+/+) controls underwent regular monitoring for their metabolic and cardiovascular phenotypes with or without MRA treatment [eplerenone (eple), 100 mg∙kg(-1) ∙day(-1) ] from 1.5 to 12.5 months of age. KEY RESULTS: Eleven months of eple treatment in obese rats (SHHF(cp/cp) eple) reduced the obesity-related metabolic disorders observed in untreated SHHF(cp/cp) rats by reducing weight gain, triglycerides and total cholesterol levels and by preserving adiponectinaemia. The MRA treatment predominantly preserved diastolic and systolic functions in obese rats by alleviating the eccentric cardiac hypertrophy observed in untreated SHHF(cp/cp) animals and preserving ejection fraction (70 ± 1 vs. 59 ± 1%). The MRA also improved survival independently of these pressure effects. CONCLUSION AND IMPLICATIONS: Early chronic eple treatment resulted in a delay in cardiac remodelling and HF onset in both SHHF(+/+) and SHHF(cp/cp) rats, whereas SHHF(cp/cp) rats further benefited from the MRA treatment through a reduction in their obesity and dyslipidaemia. These findings suggest that preventive MRA therapy may provide greater benefits in obese patients with additional risk factors of developing cardiovascular complications.

Retraction: miR-661 expression in SNAI1-induced epithelial to mesenchymal transition contributes to breast cancer cell invasion by targeting Nectin-1 and StarD10 messengers.

At the request of the University of Luxembourg and following an external investigation, the Editor and Publisher have agreed to retract this paper owing to unreliable data.

Identification of SOCS2 and SOCS6 as biomarkers in human colorectal cancer.

BACKGROUND: Over the past years, some members of the family of suppressor of cytokine signalling (SOCS) proteins have emerged as potential tumour suppressors. This study aimed at investigating the clinical significance of SOCS proteins in colorectal carcinoma (CRC). METHODS: We integrated publicly available microarray expression data on CRC in humans, analysed the expression pattern of SOCSs and assessed the predictive power of SOCS2 and SOCS6 for diagnostic purposes by generating receiver operating characteristic curves. Using laser microdissected patient material we assessed SOCS expression on RNA and protein levels as well as their methylation status in an independent CRC patient cohort. Finally, we investigated the prognostic value of SOCS2 and SOCS6. RESULTS: The meta-analysis as well as the independent patient cohort analysis reveal a stage-independent downregulation of SOCS2 and SOCS6 and identify both molecules as diagnostic biomarkers for CRC. We demonstrate a different methylation pattern within the SOCS2 promoter between tumour tissue and normal control tissue in 25% of CRC patients. Furthermore, early CRC stage patients with low expression of SOCS2 display significantly shorter disease-free survival. CONCLUSIONS: Our data offers evidence that SOCS2 and SOCS6 levels are reduced in CRC and may serve as diagnostic biomarkers for CRC patients.

miR-661 expression in SNAI1-induced epithelial to mesenchymal transition contributes to breast cancer cell invasion by targeting Nectin-1 and StarD10 messengers.

Epithelial to mesenchymal transition (EMT) is a key step toward metastasis. MCF7 breast cancer cells conditionally expressing the EMT master regulator SNAI1 were used to identify early expressed microRNAs (miRNAs) and their targets that may contribute to the EMT process. Potential targets of miRNAs were identified by matching lists of in silico predicted targets and of inversely expressed mRNAs. MiRNAs were ranked based on the number of predicted hits, highlighting miR-661, a miRNA with so far no reported role in EMT. MiR-661 was found required for efficient invasion of breast cancer cells by destabilizing two of its predicted mRNA targets, the cell-cell adhesion protein Nectin-1 and the lipid transferase StarD10, resulting, in turn, in the downregulation of epithelial markers. Reexpression of Nectin-1 or StarD10 lacking the 3'-untranslated region counteracted SNAI1-induced invasion. Importantly, analysis of public transcriptomic data from a cohort of 295 well-characterized breast tumor specimen revealed that expression of StarD10 is highly associated with markers of luminal subtypes whereas its loss negatively correlated with the EMT-related, basal-like subtype. Collectively, our non-a priori approach revealed a nonpredicted link between SNAI1-triggered EMT and the down-regulation of Nectin-1 and StarD10 through the up-regulation of miR-661, which may contribute to the invasion of breast cancer cells and poor disease outcome.

Time-resolved analysis of transcriptional events during SNAI1-triggered epithelial to mesenchymal transition.

The transcription regulator SNAI1 triggers a transcriptional program leading to epithelial to mesenchymal transition (EMT), providing epithelial cells with mesenchymal features and invasive properties during embryonic development and tumor progression. To identify early transcriptional changes occurring during SNAI1-induced EMT, we performed a time-resolved genome-scale study using human breast carcinoma cells conditionally expressing SNAI1. The approach we developed for microarray data analysis, allowed identifying three distinct EMT stages and the temporal classification of genes. Importantly, we identified unexpected, biphasic expression profiles of EMT-associated genes, supporting their pivotal role during this process. Finally, we established early EMT gene networks by identifying transcription factors and their potential targets which may orchestrate early events of EMT. Collectively, our work provides a framework for the identification and future systematic analysis of novel genes which contribute to SNAI1-triggered EMT.

A calreticulin-like molecule from the human hookworm Necator americanus interacts with C1q and the cytoplasmic signalling domains of some integrins.

Calreticulin was recently identified as a hookworm (Necator americanus) allergen, implying secretion, and contact with cells of the immune system, or significant worm attrition in the tissues of the host. As human calreticulin has been shown to bind to and neutralize the haemolytic activity of the complement component C1q, and to be putatively involved in integrin-mediated intracellular signalling events in platelets, it was of interest to determine whether a calreticulin from a successful nematode parasite of humans, with known immune modulatory and antihaemostatic properties, exhibited a capacity to interfere with complement activation and to interact with integrin domains associated with cell signalling in platelets and other leucocytes. We can now report that recombinant calreticulin failed to demonstrate significant calcium binding capacity, which is a hallmark of calreticulins in general and may indicate inappropriate folding following expression in a prokaryote. Nevertheless, recombinant calreticulin retained sufficient molecular architecture to bind to, and inhibit the haemolytic capacity of, human C1q. Furthermore, recombinant calreticulin reacted in surface plasmon resonance analysis (SPR) with peptides corresponding to cytoplasmic signalling domains of the integrins alphaIIb and alpha5, in a calcium independent manner. SPR was also used to ratify the specificity of a polyclonal antibody to hookworm calreticulin, which was then used to assess the stage specificity of expression of the native molecule (in comparison with reverse transcriptase-polymerase chain reaction), to indicate its apparent secretion, and to purify native calreticulin from worm extracts by affinity chromatography. This development will allow the functional tests described above to be repeated for native calreticulin, to ascertain its role in the host-parasite relationship.

Beta2-glycoprotein I binding to platelet microparticle membrane specifically reduces immunoreactivity of glycoproteins IIb/IIIa.

We have investigated beta2-glycoprotein I (beta2GPI) binding to platelet-derived microparticles (PMP) and its effect on GPIIb/IIIa. PMP were isolated from washed human platelets after stimulation with A23187, and analyzed by surface plasmon resonance spectroscopy. Beta2GPI as well as activated protein C (APC) or annexin V bound to PMP-coated sensorchips, demonstrating exposure of anionic phospholipids on immobilized PMP. Beta2GPI binding was impaired by calcium and occurred in a concentration-dependent manner with apparent k(on) = 2.6 x 10(4) M(-1) s(-1) and k(off) = 4.4 x 10(-3) s(-1), corresponding to a KD value of 1.7 x 10(-7) M. When analyzed by flow cytometry, the binding of certain mAbs specific for GPIIb and/or GPIIIa was reduced in the presence of beta2GPI but not of APC or annexin V, whereas the binding of anti-GPIb or anti-P-selectin mAbs, or of soluble fibrinogen remained unchanged. These results suggest a broad but specific influence of beta2GPI on GPIIb/IIIa immunoreactivity, and indicate that beta2GPI may act as a modulator of GPIIb/IIIa-dependent functions of PMP.

Cloning of an isoform of integrin-linked kinase (ILK) that is upregulated in HT-144 melanoma cells following TGF-beta1 stimulation.

We have shown previously that integrin-linked kinase (ILK) is upregulated in human HT-144 melanoma cells following TGF-beta1 stimulation. Using mRNA from TGF-beta1 stimulated HT-144 cells and reverse transcriptase polymerase chain reaction, we have isolated a cDNA encoding a protein highly homologous to ILK. Sequencing of the full-length 1359 base pair cDNA and polypeptide translation revealed that this protein, designated ILK-2, differs from the known ILK (hereafter called ILK-1) by only four amino acids, while the cDNA sequence diverges by 102 nucleotides, thus excluding that ILK-2 is an allelic variant of ILK-1. Expression of ILK-2 mRNA was observed in metastatic human HT-144 melanoma and HT-1080 fibrosarcoma cell lines, but not in normal human tissues. Moreover, stimulation of HT-144 cells with TGF-beta1, but not with EGF, PDGF-AB or insulin, induced a selective overexpression of ILK-2 mRNA as compared to ILK-1 mRNA. Bacterially-expressed GST/ILK-2 autophosphorylated and labeled myelin basic protein as well as a recombinant GST/beta3 integrin cytoplasmic tail peptide. Transfection of either ILK-2 or ILK-1 cDNA into the non-metastatic melanoma cell line SK-Mel-2, expressing exclusively ILK-1, induced anchorage independent cell growth and cell proliferation, as demonstrated by growth in soft agar. Our data provide evidence that ILK-2 is a new isoform of ILK-1 that is expressed in some highly invasive tumor cell lines but not in normal adult human tissues and whose expression is regulated by TGF-beta1.

Activation of B-Raf and regulation of the mitogen-activated protein kinase pathway by the G(o) alpha chain.

Many receptors coupled to the pertussis toxin-sensitive G(i/o) proteins stimulate the mitogen-activated protein kinase (MAPK) pathway. The role of the alpha chains of these G proteins in MAPK activation is poorly understood. We investigated the ability of Galpha(o) to regulate MAPK activity by transient expression of the activated mutant Galpha(o)-Q205L in Chinese hamster ovary cells. Galpha(o)-Q205L was not sufficient to activate MAPK but greatly enhanced the response to the epidermal growth factor (EGF) receptor. This effect was not associated with changes in the state of tyrosine phosphorylation of the EGF receptor. Galpha(o)-Q205L also potentiated MAPK stimulation by activated Ras. In Chinese hamster ovary cells, EGF receptors activate B-Raf but not Raf-1 or A-Raf. We found that expression of activated Galpha(o) stimulated B-Raf activity independently of the activation of the EGF receptor or Ras. Inactivation of protein kinase C and inhibition of phosphatidylinositol-3 kinase abolished both B-Raf activation and EGF receptor-dependent MAPK stimulation by Galpha(o). Moreover, Galpha(o)-Q205L failed to affect MAPK activation by fibroblast growth factor receptors, which stimulate Raf-1 and A-Raf but not B-Raf activity. These results suggest that Galpha(o) can regulate the MAPK pathway by activating B-Raf through a mechanism that requires a concomitant signal from tyrosine kinase receptors or Ras to efficiently stimulate MAPK activity. Further experiments showed that receptor-mediated activation of Galpha(o) caused a B-Raf response similar to that observed after expression of the mutant subunit. The finding that Galpha(o) induces Ras-independent and protein kinase C- and phosphatidylinositol-3 kinase-dependent activation of B-Raf and conditionally stimulates MAPK activity provides direct evidence for intracellular signals connecting this G protein subunit to the MAPK pathway.

Interaction between the cytodomains of the alpha 3 and beta 1 integrin subunits regulates remodelling of adhesion complexes on laminin.

The first step of laminin 1-induced signal transduction is initiated by the formation of alpha 6 beta 1 integrin-specific adhesion complexes. In contrast, on other laminin isoforms the adhesion complexes are alpha 3 beta 1 integrin-specific due to a transdominant regulation of the alpha 6 beta 1 integrin by the alpha 3 beta 1 integrin. To determine the mechanism of this regulation, peptides representing the cytoplasmic domain of the alpha 3 or alpha 6 integrin subunits were microinjected together with recombinant enhanced green fluorescence protein into live fibroblasts. Microinjection of the alpha 3 integrin peptide to laminin 1-adherent cells displaying alpha 6 beta 1 integrin-specific adhesion complexes resulted in the disengagement of the alpha 6 beta 1 integrin, while microinjection of green fluorescence protein alone or in combination with the alpha 6 integrin cytodomain had no effect. Further surface plasmon resonance studies revealed that the cytodomain of the beta 1 integrin subunit interacts with low affinity with the cytoplasmic tail of the alpha 3 integrin subunit, but not with that of several other alpha subunits including alpha 6. These results imply that the cytoplasmic tails of the integrin alpha subunits play a critical role in the regulation of integrin-induced signal transduction. In particular, the intracellular tail of the alpha 3 integrin subunit controls the formation of adhesion complexes in cells adhering to laminins.

Autocrine TGF-beta-regulated expression of adhesion receptors and integrin-linked kinase in HT-144 melanoma cells correlates with their metastatic phenotype.

We have previously shown that 2 human melanoma cell lines, the metastatic HT-144 and the non-metastatic SK-Mel-2 cells, exhibit marked in vitro heterogeneity with respect to integrin expression, migration and invasion potential. Here, we provide evidence that HT-144 melanoma cells, but not SK-Mel-2 cells, undergo a reversible transition to a fibroblastoid morphology following treatment with either their own serum-free acidified conditioned medium or biologically active exogenous TGF-beta1, thus identifying TGF-beta as an autocrine regulator of the spindle shape morphology of HT-144 melanoma cells. The fibroblastoid phenotype correlated with up-regulated beta1 and beta3 integrin and down-regulated E-cadherin expression, as shown by flow cytometry, Western blot and RT-PCR, as well as up-regulated expression of the matrix metalloproteinase MMP-9, as demonstrated by zymography. Our data further illustrate the TGF-beta1-dependent up-regulation of integrin-linked kinase and the nuclear translocation of beta-catenin, 2 intracellular proteins involved in integrin and cadherin signaling.

Both kinetic data and epitope mapping provide clues for understanding the anti-coagulant effect of five murine monoclonal antibodies to human beta2-glycoprotein I.

The interaction between five murine monoclonal antibodies (mAb) and beta2-glycoprotein I (beta2GPI) in the absence of phospholipids was studied using surface plasmon resonance-based biosensor technology. Two separate epitope regions were confirmed for the five mAb but epitopes of two mAb were shown to be overlapping but not identical. The characteristics of binding on both immobilized beta2GPI, using different chemistries of coupling to a dextran matrix and antibody surfaces prepared by two strategies of immobilization, were compared. Binding was strongly influenced by the orientation of the immobilized partner, and the five mAb showed heterogeneity in their binding to immobilized and soluble beta2GPI. The observed stoichiometries of mAb-beta2GPI complexes and the detailed analysis of the kinetics of the association and dissociation phases of the interactions with soluble and immobilized beta2GPI revealed differences in the dissociation rate constants, resulting in a 10-fold higher affinity for immobilized beta2GPI compared to soluble beta2GPI for four out of five mAb. This suggests bivalent binding of these mAb to immobilized beta2GPI. In addition, the kinetic data helped explain the differing anti-coagulant properties of these mAb.

Divalent cations differentially regulate integrin alphaIIb cytoplasmic tail binding to beta3 and to calcium- and integrin-binding protein.

We have used recombinant or synthetic alphaIIb and beta3 integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance. alpha.beta heterodimerization occurred in a 1:1 stoichiometry with a weak KD in the micromolar range. Divalent cations were not required for this association but stabilized the alpha.beta complex by decreasing the dissociation rate. alpha.beta complexation was impaired by the R995A substitution or the KVGFFKR deletion in alphaIIb but not by the beta3 S752P mutation. Recombinant calcium- and integrin-binding protein (CIB), an alphaIIb-specific ligand, bound to the alphaIIb cytoplasmic peptide in a Ca2+- or Mn2+-independent, one-to-one reaction with a KD value of 12 microM. In contrast, in vitro liquid phase binding of CIB to intact alphaIIbbeta3 occurred preferentially with Mn2+-activated alphaIIbbeta3 conformers, as demonstrated by enhanced coimmunoprecipitation of CIB with PAC-1-captured Mn2+-activated alphaIIbbeta3, suggesting that Mn2+ activation of intact alphaIIbbeta3 induces the exposure of a CIB-binding site, spontaneously exposed by the free alphaIIb peptide. Since CIB did not stimulate PAC-1 binding to inactive alphaIIbbeta3 nor prevented activated alphaIIbbeta3 occupancy by PAC-1, we conclude that CIB does not regulate alphaIIbbeta3 inside-out signaling, but rather is involved in an alphaIIbbeta3 post-receptor occupancy event.

Activating mutations of the Gs alpha gene are associated with low levels of Gs alpha protein in growth hormone-secreting tumors.

Evidence suggests the existence of a direct relationship between cellular Gs alpha content and activation of the adenylyl cyclase system. Data on Gs alpha levels in endocrine tumors that depend on cAMP for growth, particularly pituitary adenomas, are still limited. The levels of Gs alpha protein were evaluated in 11 GH-secreting adenomas with Gs alpha mutations (gsp+) and 15 without (gsp). Complementary DNAs from gsp+ tumors contained very low amounts of wild-type Gs alpha sequences, indicating a preponderance of the mutant Gs alpha transcripts in these tumors. Immunoblotting of Gs alpha protein showed that the two isoforms were present at high levels in all gsp-, but were undetectable or barely detectable in gsp+. The low Gs alpha content in gsp+ tumors was not due to a reduction in ribonucleic acid synthesis or stability, as Gs alpha messenger ribonucleic acid levels were similar in wild-type and mutant tissues. Treatment of gsp- cells with cholera toxin caused a marked reduction of Gs alpha levels. As in other cell systems cholera toxin increases Gs alpha degradation, our data are consistent with an accelerated removal of mutant Gs alpha. This may represent an additional mechanism of feedback response to the constitutive activation of cAMP signaling in pituitary tumors with mutations in the Gs alpha gene.

Immunopurification of human beta2-glycorprotein I with a monoclonal antibody selected for its binding kinetics using a surface plasmon resonance biosensor.

The beta2-glycoprotein I (beta2GPI)-binding properties of five murine monoclonal antibodies immobilized as capture antibodies were studied using surface plasmon resonance detection. The monoclonal antibody with the fastest dissociation kinetics (6F3) was selected for the development of an immunoaffinity chromatography procedure, assuming that its behaviour would be similar in both systems since the covalent coupling chemistries involved amino groups in both cases. Under our experimental conditions of a fast one-step procedure, beta2GPI was purified to homogeneity from human plasma with a yield of about 50%. Beta2GPI was eluted under fairly mild conditions, either at low pH or at high pH. The immunoadsorbent was used five times without any apparent loss of binding capacity. The immunopurified protein showed similar binding to cardiolipin-coated polystyrene wells as beta2GPI purified by conventional methods. However, differences in the pattern of immunoreactivity in relation to the purification procedure were observed by surface plasmon resonance using the monoclonal antibody with the highest association kinetics (9G1) immobilized on the sensor surface.

Oncogenic role of heterotrimeric G proteins.

Mutations that constitutively activate the alpha chains of Gs and Gi2 by inhibiting their intrinsic GTPase activity are present in human endocrine tumours. The gsp oncogene is mainly found in pituitary GH secreting tumours and thyroid hyperfunctioning adenomas, where it induces a constitutive activation of the adenylyl cyclase-cAMP pathway. In pituitary and thyroid cells, this signal leads to abnormal proliferation and a persistent activation of differentiated functions. The gip2 oncogene has been identified in tumours of the ovary and adrenal cortex. Although the mechanisms of the oncogenic action of mutationally activated alpha i2 are less clear than those of alpha s, the protein can induce transformation of certain cell types. At least five other alpha chains, which share with alpha s and alpha i2 common structural and functional mechanisms of GTP hydrolysis, activate mitogenic pathways leading to transformation. In addition, the G protein beta gamma subunits clearly control signals involved in cell growth. So far, there is no evidence for mutations of these molecules in human tumours. Further studies will tell us whether at present we know of only two members of a much larger family of G protein oncogenes.

Regenerated cellulose-based hemodialyzers with immobilized proteins as potential devices for extracorporeal immunoadsorption procedures: an assessment of protein coupling capacity and in vitro dialysis performances.

The development of immunoadsorbents usable with whole blood should offer the potential for making significant improvements in extracorporeal immunoadsorption procedures. In contrast to traditional chromatographic media, these hemocompatible matrices could be used without requiring the previous step of the separation of blood cells and plasma. Conventional hemodialyzers seem to be particularly appropriate for such a purpose. This paper describes a feasibility study of the preparation of immunoaffinity supports, from regenerated cellulose (Cuprophan)-based dialyzers by cyanogen bromide activation and coupling of bovine serum albumin or human immunoglobin G, used as models for immunochemical ligands. Several parameters of the activation and coupling steps were studied in order to define the optimum preparation conditions. In addition, the preservation of the transport properties (clearance and ultrafiltration) of the modified hemodialyzers was evaluated in vitro to ensure that the device potentially could be used in future human therapeutic applications with no risk of massive removal of solutes and fluid from the blood. Results indicate that 150-300 mg of immunoglobulins can be immobilized per square meter of Cuprophan. Modified hemodialyzers show a slight decrease of their clearance values and a slight increase of their ultrafiltration coefficients, and thus they can be proposed as reliable carrier material for extracorporeal cleansing systems.

Activation of cyclic nucleotide phosphodiesterases in FRTL-5 thyroid cells expressing a constitutively active Gs alpha.

The expression of a constitutively activated Gs alpha protein in the rat thyroid cell line FRTL-5 causes an increase in the hormone-independent adenylyl cyclase activity and promotes TSH-independent growth of the cells. In spite of the constitutive activation of the adenylyl cyclase, the basal cAMP levels in these cells are only marginally increased. To define the role of phosphodiesterases (PDEs) in the genesis of this phenotype, cyclic nucleotide hydrolysis was determined in two cell lines expressing a mutated Gs alpha (Q227L). In these cells, the hydrolysis of both cAMP and cGMP was markedly increased in comparison with normal cells. This increase is the result of the activation of different forms of PDEs. Analysis of the cGMP hydrolysis and Ca++/calmodulin stimulation of the PDE activity indicated that the activity of a Ca++/calmodulin-stimulated PDE is increased in both cell lines. In addition, an increase in high-affinity, rolipram-sensitive cAMP-PDE activity was associated in both cell lines with the appearance of a 67-68 kilodalton (kDa) protein that cross-reacts with two antibodies against cAMP-PDEs. This form had the properties of ratPDE3.2/PDE4D2, a cAMP-PDE that is inducible by TSH in wild type cells. That an increase in cAMP-specific, rolipram-sensitive PDE plays a role in the phenotype induced by Q227L Gs alpha was confirmed by measurements of the mitogenic activity. Incubation with rolipram, which had no effect on wild type cells, caused an increase in cAMP levels and further stimulated TSH-independent proliferation in both cell lines carrying the mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoadsorption procedure as a potential method for the specific beta 2-microglobulin removal from plasma of patients with chronic renal failure.

beta 2-Microglobulin (beta 2-M), which accumulates in the plasma of patients undergoing long-term dialysis, has been identified as the principal precursor protein of amyloid fibrils in dialysis-related amyloidosis. As no specific treatment for this affection has been yet established, an extracorporeal immunoadsorption procedure appears to be an attractive therapeutic approach to remove beta 2-M. Several murine monoclonal antibodies to human beta 2-M were developed and compared as affinity ligands. One of them was selected on the basis of its specificity and adsorption capacity. In order to achieve maximum efficiency in protein removal, different parameters of the procedure were studied and optimized: effect of antibody coupling density, determination of maximum adsorption capacity of the immunoadsorbents and influence of antigen concentration and of flow-rate on antigen capture efficiency. The conditions of regeneration of immunoaffinity sorbents were also investigated to allow their multiple use without loss of adsorption capacity. The results show the validity of the proposed technique in removing beta-M from plasma of patients with chronic renal failure.

Expression of mutationally activated G alpha s stimulates growth and differentiation of thyroid FRTL5 cells.

The alpha subunit of the GTP-binding protein Gs mediates hormonal stimulation of adenylyl cyclase. Human pituitary and thyroid tumours harbour mutations of G alpha s that constitutively activate the protein by inhibiting its intrinsic GTPase activity. We have investigated the mitogenic action of mutationally activated alpha s in thyroid FRTL5 cells, a cell line dependent upon thyroid-stimulating hormone (TSH) for both growth and differentiation. Introduction of alpha s carrying the substitution of glutamine-227 with leucine (Q227L alpha s) by retroviral infection of FRTL5 cells resulted in the expected stimulation of membrane adenylyl cyclase activity and in increased intracellular accumulation of cAMP. Measurements of cytosolic Ca2+ levels did not detect any concomitant effect on the polyphosphoinositide-Ca2+ signalling pathway. Expression of Q227L alpha s conferred to FRTL5 cells the ability to synthesize DNA in the absence of TSH, as revealed by [3H]thymidine incorporation experiments, and to proliferate independently of the mitogenic hormone, although with a rate of growth slower than that observed with TSH stimulation. The effect of Q227L alpha s on cell proliferation was associated with the constitutive activation of iodide uptake. The results indicate that expression of mutationally activated G alpha s is sufficient to bypass the requirement for TSH and promotes autonomous growth and activation of thyroid-specific differentiated functions in FRTL5 cells.