T-bet controls intestinal mucosa immune responses via repression of type 2 innate lymphoid cell function.

Innate lymphoid cells (ILCs) play an important role in regulating immune responses at mucosal surfaces. The transcription factor T-bet is crucial for the function of ILC1s and NCR+ ILC3s and constitutive deletion of T-bet prevents the development of these subsets. Lack of T-bet in the absence of an adaptive immune system causes microbiota-dependent colitis to occur due to aberrant ILC3 responses. Thus, T-bet expression in the innate immune system has been considered to dampen pathogenic immune responses. Here, we show that T-bet plays an unexpected role in negatively regulating innate type 2 responses, in the context of an otherwise intact immune system. Selective loss of T-bet in ILCs leads to the expansion and increased activity of ILC2s, which has a functionally important impact on mucosal immunity, including enhanced protection from Trichinella spiralis infection and inflammatory colitis. Mechanistically, we show that T-bet controls the intestinal ILC pool through regulation of IL-7 receptor signalling. These data demonstrate that T-bet expression in ILCs acts as the key transcriptional checkpoint in regulating pathogenic vs. protective mucosal immune responses, which has significant implications for the understanding of the pathogenesis of inflammatory bowel diseases and intestinal infections.

Blocking antibodies induced by allergen-specific immunotherapy ameliorate allergic airway disease in a human/mouse chimeric model.

BACKGROUND: Allergen-specific immunotherapy (AIT) induces specific blocking antibodies (Ab), which are claimed to prevent IgE-mediated reactions to allergens. Additionally, AIT modulates cellular responses to allergens, for example, by desensitizing effector cells, inducing regulatory T and B lymphocytes and immune deviation. It is still enigmatic which of these mechanisms mediate(s) clinical tolerance. We sought to address the role of AIT-induced blocking Ab separately from cellular responses in a chimeric human/mouse model of respiratory allergy. METHODS: Nonobese diabetic severe combined immunodeficient γc-/- (NSG) mice received intraperitoneally allergen-reactive PBMC from birch pollen-allergic patients together with birch pollen extract and human IL-4. Engraftment was assessed by flow cytometry. Airway hyperresponsiveness (AHR) and bronchial inflammation were analyzed after intranasal challenges with allergen or PBS. Sera collected from patients before and during AIT with birch pollen were added to the allergen prior to intranasal challenge. The IgE-blocking activity of post-AIT sera was assessed in vitro. RESULTS: Human cells were detected in cell suspensions of murine lungs and spleens indicating successful humanization. Humanized mice displayed a more pronounced AHR and bronchial inflammation when challenged with allergen compared to negative controls. Post-AIT sera exerted IgE-blocking activity. In contrast to pre-AIT sera, the presence of heterologous and autologous post-AIT sera significantly reduced the allergic airway inflammation and matched their IgE-blocking activity determined in vitro. CONCLUSION: Our data demonstrate that post-AIT sera with IgE-blocking activity ameliorate allergic airway inflammation in a human/mouse chimeric model of respiratory allergy independently of AIT-induced cellular changes.

O-GlcNAcylation of STAT5 controls tyrosine phosphorylation and oncogenic transcription in STAT5-dependent malignancies.

The signal transducer and activator of transcription 5 (STAT5) regulates differentiation, survival, proliferation and transformation of hematopoietic cells. Upon cytokine stimulation, STAT5 tyrosine phosphorylation (pYSTAT5) is transient, while in diverse neoplastic cells persistent overexpression and enhanced pYSTAT5 are frequently found. Post-translational modifications might contribute to enhanced STAT5 activation in the context of transformation, but the strength and duration of pYSTAT5 are incompletely understood. We found that O-GlcNAcylation and tyrosine phosphorylation act together to trigger pYSTAT5 levels and oncogenic transcription in neoplastic cells. The expression of a mutated hyperactive gain-of-function (GOF) STAT5 without O-GlcNAcylation resulted in decreased tyrosine phosphorylation, oligomerization and transactivation potential and complete loss of oncogenic transformation capacity. The lack of O-GlcNAcylation diminished phospho-ERK and phospho-AKT levels. Our data show that O-GlcNAcylation of STAT5 is an important process that contributes to oncogenic transcription through enhanced STAT5 tyrosine phosphorylation and oligomerization driving myeloid transformation. O-GlcNAcylation of STAT5 could be required for nutrient sensing and metabolism of cancer cells.

MYC selects against reduced BCL2A1/A1 protein expression during B cell lymphomagenesis.

Rearrangements of MYC or ABL proto-oncogenes lead to deregulated expression of key-regulators of cell cycle and cell survival, thereby constituting important drivers of blood cancer. Members of the BCL-2 family of apoptosis regulators contribute to oncogenic transformation downstream of these oncogenes, but the role of anti-apoptotic BCL2A1/A1 in transformation and drug resistance caused by deregulation of these oncogenes remains enigmatic. Here we analyzed the role of A1 in MYC as well as ABL kinase-driven blood cancer in mice, employing in vivo RNAi. We report that overexpression of either oncogene leads to a significant increase in A1 protein levels in otherwise A1-negative B cell progenitors, indicating a key role downstream of these oncogenes to secure survival during transformation. Knockdown of A1 by RNAi, however, did not impact on tumor latency in v-Abl-driven pre-B-ALL. In contrast, A1 knockdown in premalignant Eµ-MYC mice caused a significant reduction of transgenic pre-B cells without impacting on tumor latency as the emerging lymphomas escaped silencing of A1 expression. These findings identify A1 as a MYC target that can be induced prematurely during B cell development to aid expansion of otherwise cell-death-prone MYC transgenic pre-B cells. Hence, A1 should be considered as a putative drug target in MYC-driven blood cancer.

CDK6-a review of the past and a glimpse into the future: from cell-cycle control to transcriptional regulation.

The G1 cell-cycle kinase CDK6 has long been thought of as a redundant homolog of CDK4. Although the two kinases have very similar roles in cell-cycle progression, it has recently become apparent that they differ in tissue-specific functions and contribute differently to tumor development. CDK6 is directly involved in transcription in tumor cells and in hematopoietic stem cells. These functions point to a role of CDK6 in tissue homeostasis and differentiation that is partially independent of CDK6's kinase activity and is not shared with CDK4. We review the literature on the contribution of CDK6 to transcription in an attempt to link the new findings on CDK6's transcriptional activity to cell-cycle progression. Finally, we note that anticancer therapies based on the inhibition of CDK6 kinase activity fail to take into account its kinase-independent role in tumor development.

Stat5 gene dosage in T cells modulates CD8+ T-cell homeostasis and attenuates contact hypersensitivity response in mice.

BACKGROUND: Contact hypersensitivity assay (CHS) faithfully models human allergies. The Stat5 transcription factors are essential for both lymphocyte development and acute immune responses. Although consequences of Stat5 ablation and transgenic overexpression for the lymphocyte development and functions have been extensively studied, the role of Stat5 gene dosage in contact allergies has not been addressed. OBJECTIVE: We investigated the effect of Stat5 gene dosage modulation in contact allergies using CHS in mice. METHODS: Transgenic animals heterozygous for the germline Stat5 null allele were subjected to CHS. To dissect cell type sensitive to Stat5 gene dosage, animals with Stat5 haplo-insufficiency in T cells, where one Stat5 allele was removed by Lck-Cre-mediated deletion (Stat5(ΔT/+)), were tested by CHS. Frequency of T cells, B cells, and monocytes were analyzed in Stat5(ΔT/+) and wild-type animals by flow cytometry. Proliferation of Stat5(ΔT/+) CD8(+) T cells was studied in vitro by stimulation with IL-4 and IL-2 cytokines, and changes in the expression of Stat5 target genes were assayed by quantitative real-time PCR assay. RESULT: Haplo-insufficiency of Stat5 in T cells leads to the reduction in CD8(+) T cells in all lymphoid organs and attenuates CHS response. Stat5(ΔT/+) CD8(+) T cells failed to fully activate Stat5-dependent expression of cell cycle/survival target genes, such as Bcl2 and Pim1, and to proliferate efficiently in response to IL-2 and IL-4 cytokine. CONCLUSION: Our data identify Stat5 as a dose-dependent regulator of CD8(+) T-cell functions in contact allergies and suggest that modulation of Stat5 dosage could be used to target contact allergies in humans.

NK cell development in bone marrow and liver: site matters.

The NKp46 protein is found on resting and activated natural killer (NK) cells and is involved in the recognition of malignant and infected cells. The expression of NKp46 is believed to precede that of DX5 in early NK cell development. We show that this is not the case in the bone marrow (BM). Here, NKp46 is predominantly expressed after DX5, whereas the liver harbors a subpopulation that expresses NKp46 but not DX5. NK cell precursors in the liver show much lower levels of Eomesodermin than NK cell precursors in the BM, although they express higher levels of granzymes and unlike the NK cell precursors in the BM are fully able to degranulate and produce interferon gamma (IFN-γ). The development of NK cells thus differs between the two organs. This needs to be considered when using NKp46 and DX5 as NK cell markers and when performing NK cell-specific gene deletion in Ncr1 transgenic mice.

PAK-dependent STAT5 serine phosphorylation is required for BCR-ABL-induced leukemogenesis.

The transcription factor STAT5 (signal transducer and activator of transcription 5) is frequently activated in hematological malignancies and represents an essential signaling node downstream of the BCR-ABL oncogene. STAT5 can be phosphorylated at three positions, on a tyrosine and on the two serines S725 and S779. We have investigated the importance of STAT5 serine phosphorylation for BCR-ABL-induced leukemogenesis. In cultured bone marrow cells, expression of a STAT5 mutant lacking the S725 and S779 phosphorylation sites (STAT5(SASA)) prohibits transformation and induces apoptosis. Accordingly, STAT5(SASA) BCR-ABL(+) cells display a strongly reduced leukemic potential in vivo, predominantly caused by loss of S779 phosphorylation that prevents the nuclear translocation of STAT5. Three distinct lines of evidence indicate that S779 is phosphorylated by group I p21-activated kinase (PAK). We show further that PAK-dependent serine phosphorylation of STAT5 is unaffected by BCR-ABL tyrosine kinase inhibitor treatment. Interfering with STAT5 phosphorylation could thus be a novel therapeutic approach to target BCR-ABL-induced malignancies.

JunB is a gatekeeper for B-lymphoid leukemia.

Loss of JunB has been observed in human leukemia and lymphoma, but it remains unknown, whether this loss is relevant to disease progression. Here, we investigated the consequences of JunB deficiency using Abelson-induced B-lymphoid leukemia as a model system. Mice deficient in JunB expression succumbed to Abelson-induced leukemia with increased incidence and significantly reduced latency. Similarly, bcr/abl p185-transformed JunB-deficient (junB(Delta/Delta)) cells induced leukemia in RAG2(-/-) mice displaying a more malignant phenotype. These observations indicated that cell intrinsic effects within the junB(Delta/Delta) tumor cells accounted for the accelerated leukemia development. Indeed, explantated bcr/abl p185 transformed junB(Delta/Delta) cells proliferated faster than the control cells. The proliferative advantage emerged slowly after the initial transformation process and was associated with increased expression levels of the cell cycle kinase cdk6 and with decreased levels of the cell cycle inhibitor p16(INK4a). These alterations were due to irreversible reprogramming of the cell, because - once established - accelerated disease induced by junB(Delta/Delta) cells was not reverted by re-introducing JunB. Consistent with this observation, we found that the p16 promoter was methylated. Thus, JunB functions as a gatekeeper during tumor evolution. In its absence, transformed leukemic cells acquire an enhanced proliferative capacity, which presages a more malignant disease.

Stat5a/b contribute to interleukin 7-induced B-cell precursor expansion, but abl- and bcr/abl-induced transformation are independent of stat5.

The cytokines interleukin 7 (IL-7) and interleukin 4 (IL-4) regulate lymphoid differentiation and function and activate the transcription factor Stat5. Using mice deficient for the 2 highly related transcription factors, Stat5a and Stat5b (Stat5a/b(-/-)), we investigated the role of Stat5 for B-cell differentiation, expansion, and function. Peripheral blood B cells of Stat5-deficient mice are significantly reduced, but no proliferation defects in response to various mitogenic stimuli are found. Also, IgM and IgG1 antibody production and immunoglobulin class switching are not affected. Pre- and pro-B cells of Stat5-deficient animals were found to have reduced responses to IL-7. Pro- and pre-B cells are the target cells of the abl oncogene and numerous studies have suggested that Stat5a/b is essential for transformation by derivatives of the Abelson (abl) gene. To assess the role of Stat5a/b in transformation, we have evaluated the ability of various abl derivatives to transform cells from Stat5a/b-deficient mice in vitro or in vivo. We demonstrate that the absence of Stat5a/b is not essential for the induction of lymphoid or myeloid tumors in vivo or on the ability to transform bone marrow cells in vitro.

Stat5 activation is uniquely associated with cytokine signaling in peripheral T cells.

The activation and subsequent proliferation of peripheral T cells requires the engagement of the T cell and a cytokine receptor, typically the IL-2 or IL-4 receptors. Critical to understanding the regulation of peripheral T cells is the knowledge of the unique contributions of each receptor to full T cell activation and cell cycle progression. Mice deficient in Stat5a and Stat5b have demonstrated the essential role that these highly related proteins play in cell cycle progression following peripheral T cell activation. Here we demonstrate that activation of the Stat5 proteins by tyrosine phosphorylation is uniquely contributed by cytokine receptor signaling and specifically does not occur through the T cell receptor complex.

Stat5 is required for IL-2-induced cell cycle progression of peripheral T cells.

Many cytokines activate two highly homologous Stat proteins, 5a and 5b. Mice deficient in both genes lack all growth hormone and prolactin functions but retain functions associated with cytokines such as erythropoietin. Here, we demonstrate that, while lymphoid development is normal, Stat5a/b mutant peripheral T cells are profoundly deficient in proliferation and fail to undergo cell cycle progression or to express genes controlling cell cycle progression. In addition, the mice lack NK cells, develop splenomegaly, and have T cells with an activated phenotype, phenotypes seen in IL-2 receptor beta chain-deficient mice. These phenotypes are not seen in mice lacking Stat5a or Stat5b alone. The results demonstrate that the Stat5 proteins, redundantly, are essential mediators of IL-2 signaling in T cells.

A rate limiting function of cdc25A for S phase entry inversely correlates with tyrosine dephosphorylation of Cdk2.

The cdc25A phosphatase removes inhibitory phosphates from threonine-14 and tyrosine-15 of cyclin dependent kinase-2 (cdk2) in vitro, and it is therefore widely assumed that cdc25A positively regulates cyclin E- and A-associated cdk2 activity at the G1 to S phase transition of the mammalian cell division cycle. Human cdc25A was introduced into mouse NIH3T3 fibroblasts co-expressing a form of the colony-stimulating factor-1 (CSF-1) receptor that is partially defective in transducing mitogenic signals. Cdc25A enabled these cells to form colonies in semisolid medium containing serum plus human recombinant CSF-1 in a manner reminiscent of cells rescued by c-myc. However, cdc25A-rescued cells could not proliferate in chemically defined medium containing CSF-1 and continued to require c-myc function for S phase entry. When contact-inhibited cells overexpressing cdc25A were dispersed and stimulated to synchronously enter the cell division cycle, they entered S phase 2-3 h earlier than their parental untransfected counterparts. Shortening of G1 phase temporally correlated with more rapid degradation of the cdk inhibitor p27Kip1 and with premature activation of cyclin A-dependent cdk2. Paradoxically, tyrosine phosphorylation of cdk2 increased considerably as cells entered S phase, and cdc25A overexpression potentiated rather than diminished this effect. At face value, these results are inconsistent with the hypothesis that cdc25A acts directly on cdk2 to activate its S phase promoting function.

Assembly of cyclin D-dependent kinase and titration of p27Kip1 regulated by mitogen-activated protein kinase kinase (MEK1).

A constitutively active form of mitogen-activated protein kinase kinase (MEK1) was synthesized under control of a zinc-inducible promoter in NIH 3T3 fibroblasts. Zinc treatment of serum-starved cells activated extracellular signal-regulated protein kinases (ERKs) and induced expression of cyclin D1. Newly synthesized cyclin D1 assembled with cyclin-dependent kinase-4 (CDK4) to form holoenzyme complexes that phosphorylated the retinoblastoma protein inefficiently. Activation of the MEK1/ERK pathway neither triggered degradation of the CDK inhibitor kinase inhibitory protein-1 (p27(Kip1)) nor led to activation of cyclin E- and A-dependent CDK2, and such cells did not enter the DNA synthetic (S) phase of the cell division cycle. In contrast, zinc induction of active MEK1 in cells also engineered to ectopically overexpress cyclin D1 and CDK4 subunits generated levels of cyclin D-dependent retinoblastoma protein kinase activity approximating those achieved in cells stimulated by serum. In this setting, p27(Kip1) was mobilized into complexes containing cyclin D1; cyclin E- and A-dependent CDK2 complexes were activated; and serum-starved cells entered S phase. Thus, although the activity of p27(Kip1) normally is canceled through a serum-dependent degradative process, overexpressed cyclin D1-CDK complexes sequestered p27(Kip1) and reduced the effective inhibitory threshold through a stoichiometric mechanism. A fraction of these cells completed S phase and divided, but they were unable to continuously proliferate, indicating that other serum-responsive factors ultimately became rate limiting for cell cycle progression. Therefore, the MEK/ERK pathway not only acts transcriptionally to induce the cyclin D1 gene but functions posttranslationally to regulate cyclin D1 assembly with CDK4 and to thereby help cancel p27(Kip1)-mediated inhibition.

The cyclin-dependent kinase inhibitor p21cip1 mediates the growth inhibitory effect of phorbol esters in human venous endothelial cells.

Long-term application of the phorbol ester phorbol 12,13-dibutyrate (PDBu) inhibits the proliferation of human venous endothelial cells. The cyclin-dependent kinase inhibitor p21cip1 is a potential candidate mediating the PDBu-induced delayed entry of the cells into S-phase (by approximately 10 h when compared with cells stimulated with basic fibroblast growth factor (bFGF)). Levels of p21cip1 (protein and mRNA) rapidly rise (within approximately 2 h) in endothelial cells treated with the active isomer beta-PDBu, but not with alpha-PDBu; this effect is blocked by the mitogen-activated protein kinase kinase-1 (Mek1) inhibitor PD098059 and by the protein kinase C (PKC) antagonists GF109203X and rottlerin (selective for PKC-delta), but not Gö 6976 (selective for Ca2+-dependent PKC isoforms). Rapamycin blocks the PDBu-induced accumulation of p21cip1 (but not of the cognate mRNA), indicating an action of PKC on p21(cip1) mRNA translation. If endothelial cells are recruited into the cell cycle by bFGF, p21cip1 mRNA and protein levels rise initially (within 2 h) and decline subsequently such that p21cip1 drops to a minimum prior to the initiation of DNA synthesis (i.e. after approximately 12 h). In bFGF-stimulated cells, changes in p21cip1 mRNA and protein are strictly linked. In contrast, the levels of p21cip1 mRNA decline substantially (>10 h) before the protein decreases in PDBu-stimulated cells. Thus, PKC (presumably PKC-delta) regulates the amounts of p21cip1 in endothelial cells at the level of mRNA accumulation and translation, leading to a rapid and robust induction; following persistent PKC activation, p21(cip1) remains elevated despite reduced mRNA levels, indicating an enhanced stability of the protein. The bFGF-mediated increase in p21cip1 is blocked by the Mek1 inhibitor, but not by GF109203X; hence, in endothelial cells, induction of p21cip1 by PKC- and growth factor-dependent signaling is achieved by distinct pathways that converge and require activation of the mitogen-activated protein kinase cascade. The beta-PDBu-induced delayed S-phase entry and drop in p21cip1 are reversed if GF109203X is added 4 h after beta-PDBu to prevent persistent PKC activation. These observations indicate a cause and effect relation between sustained p21cip1 elevations and the delay in S-phase entry induced by beta-PDBu.

Increase by tri-iodothyronine of endothelin-1, fibronectin and von Willebrand factor in cultured endothelial cells.

Hyperthyroidism is associated with elevated plasma levels of endothelium-derived proteins such as von Willebrand factor (vWF), fibronectin (FN) and endothelin-1 (ET-1). This study was designed to characterize the mechanisms involved in this phenomenon at the cellular level. vWF, FN and ET-1 secretion and mRNA expression were measured in human umbilical vein endothelial cells (HUVECs) exposed to tri-iodothyronine (T3) for 13 +/- 1 days, using ELISA, Western blot, RIA and Northern blot analysis respectively. Exposure of HUVECs to T3 significantly increased vWF secretion (50 ng T3/ml: 117 +/- 5%, P < 0.01; 100 ng T3/ml: 127 +/- 26%, P < 0.01) as well as vWF mRNA expression (50 ng/ml: 116 +/- 13%, P < 0.001; 100 ng/ml: 136 +/- 30%, P < 0.002) (results are means +/- S.D. analysed by the Wilcoxon signed rank test). FN secretion was significantly affected by 50 (145 +/- 42% of control, P < 0.05) and 100 (116.8 +/- 16% of control, P < 0.05) ng T3/ml, and FN mRNA expression by 50 ng T3/ml (123 +/- 20%, P < 0.05). Long-term incubation with T3 increased both ET-1 secretion (25 ng/ml: 124 +/- 25%, P < 0.001; 50 ng/ml: 165 +/- 53%, P < 0.05; 100 ng/ml: 116 +/- 17%, P < 0.05) and prepro-ET-1 mRNA expression (25 ng/ml: 112 +/- 16%, P < 0.05; 50 ng/ml: 134 +/- 43%, P < 0.02; 100 ng/ml: 120 +/- 20%, P < 0.02). Protein kinase C (PKC) isoforms epsilon and beta II were not significantly affected by T3, whereas PKC alpha was increased in whole cell lysates and in membrane fractions of cells incubated with 100 but not 50 ng T3/ml. Prepro-ET-1 mRNA stability, cell numbers and proliferation, measured by [3H]thymidine assays, remained unaffected in HUVECs after exposure to T3. These data indicate thyroid hormone-induced upregulation of mRNA expression and protein synthesis of vWF, FN and ET-1, by PKC alpha-, beta II- and epsilon-independent pathways, explaining, at least in part, increased plasma concentrations of endothelial proteins and peptides in the hyperthyroid state.

Stimulation of the mitogen-activated protein kinase via the A2A-adenosine receptor in primary human endothelial cells.

Adenosine exerts a mitogenic effect on human endothelial cells via stimulation of the A2A-adenosine receptor. This effect can also be elicited by the beta2-adrenergic receptor but is not mimicked by elevation of intracellular cAMP levels. In the present work, we report that stimulation of the A2A-adenosine receptor and of the beta2-adrenergic receptor activates mitogen-activated protein kinase (MAP kinase) in human endothelial cells based on the following criteria: adenosine analogues and beta-adrenergic agonists cause an (i) increase in tyrosine phosphorylation of the p42 isoform and to a lesser extent of the p44 isoform of MAP kinase and (ii) stimulate the phosphorylation of myelin basic protein by MAP kinase; (iii) this is accompanied by a redistribution of the enzyme to the perinuclear region. Pretreatment of the cells with cholera toxin (to down-regulate Gsalpha) abolishes activation of MAP kinase by isoproterenol but not that induced by adenosine analogues. In addition, MAP kinase stimulation via the A2A-adenosine receptor is neither impaired following pretreatment of the cells with pertussis toxin (to block Gi-dependent pathways) nor affected by GF109203X (1 microM; to inhibit typical protein kinase C isoforms) nor by a monoclonal antibody, which blocks epidermal growth factor-dependent signaling. In contrast, MAP kinase activation is blocked by PD 098059, an inhibitor of MAP kinase kinase 1 (MEK1) activation, which also blunts the A2A-adenosine receptor-mediated increase in [3H]thymidine incorporation. Activation of the A2A-adenosine receptor is associated with increased levels of GTP-bound p21(ras). Thus, our experiments define stimulation of MAP kinase as the candidate cellular target mediating the mitogenic action of the A2A-adenosine receptor on primary human endothelial cells; the signaling pathway operates via p21(ras) and MEK1 but is independent of Gi, Gs, and the typical protein kinase C isoforms. This implies an additional G protein which links this prototypical Gs-coupled receptor to the MAP kinase cascade.

High-glucose incubation of human umbilical-vein endothelial cells does not alter expression and function either of G-protein alpha-subunits or of endothelial NO synthase.

Alterations in G-protein-controlled signalling pathways (primarily pathways controlled by Gs and Gi) have been reported to occur in animal models of diabetes mellitus. We have therefore studied the effect of a long-term exposure of human umbilical vein endothelial cells to elevated concentrations of glucose on expression and function of G-protein subunits and endothelial NO synthase. Long-term incubation in high glucose (30 mM for 15 days) did not affect the levels of Gialpha-2, Gqalpha, the splice variants (long and short form) of Gsalpha, and the G-protein beta-subunits or adenylate cyclase activity; basal, as well as isoprenaline-, forskolin- and guanosine 5'-[gamma-thio]triphosphate-stimulated enzyme activities were comparable in high- and low-glucose-treated cells, thus ruling out any functional changes in the stimulatory pathway. Pretreatment of endothelial cells with pertussis toxin blocked a substantial fraction (50%) of the mitogenic response to serum factor(s) which depend(s) of functional Gi2. The sensitivity of cells cultured in high glucose was comparable with that of the paired controls maintained in normal glucose (EC50 = 3.1 +/- 0.5 and 3.3 +/- 0.4 ng/ml respectively). Similarly, we failed to detect any differences in endothelial NO synthase expression, or intracellular distribution and basal activity of the enzyme in endothelial cells cultured in high glucose. Stimulation of NO synthase in intact cells revealed a comparable response to the calcium ionophore (A23187). In contrast, stimulation with histamine (which acts via H1-receptors predominantly coupled to Gq) resulted in a significantly increased response in the cells maintained in high glucose. These data are suggestive of an altered H1-histamine receptor-Gq-phospholipase C pathway in endothelial cells cultured in high glucose concentrations, but rule out any glucose-induced functional changes in Gs- and Gi-controlled signalling pathways.