Enhanced production of IL-17A in patients with severe asthma is inhibited by 1α,25-dihydroxyvitamin D3 in a glucocorticoid-independent fashion

Background: TH17 cells are proposed to play a role in the pathology of asthma, including steroid-resistant (SR) disease. We previously identified a steroid-enhancing function of vitamin D in patients with SR asthma in restoring the impaired response to steroids for production of the anti-inflammatory cytokine IL-10. / Objective: We sought to investigate the production of the TH17-associated cytokines IL-17A and IL-22 in culture in patients with moderate-to-severe asthma defined on the basis of their clinical response to steroids and the susceptibility of this response to inhibition by steroids and the active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25[OH]2D3). / Methods: PBMCs were stimulated in culture with or without dexamethasone and 1,25(OH)2D3. A cytometric bead array, ELISA, and intracellular cytokine staining were used to assess cytokine production. The role of CD39 in inhibition of the TH17 response was studied by using quantitative real-time PCR, flow cytometry, and addition of the antagonist POM-1 to culture. / Results: Asthmatic patients synthesized much higher levels of IL-17A and IL-22 than nonasthmatic control subjects, with patients with SR asthma expressing the highest levels of IL-17A. Glucocorticoids did not inhibit IL-17A cytokine expression in patients and enhanced production in cultures from control subjects. Treatment with 1,25(OH)2D3 with or without dexamethasone significantly reduced both IL-17A and IL-22 levels. An antagonist of the ectonucleotidase CD39 reversed 1,25(OH)2D3-mediated inhibition of the IL-17A response. / Conclusion: Patients with severe asthma exhibit increased levels of TH17 cytokines, which are not inhibited by steroids. 1,25(OH)2D3 inhibits TH17 cytokine production in all patients studied, irrespective of their clinical responsiveness to steroids, identifying novel steroid-enhancing properties of vitamin D in asthmatic patients

A Concerted Kinase Interplay Identifies PPARγ as a Molecular Target of Ghrelin Signaling in Macrophages

The peroxisome proliferator-activator receptor PPARγ plays an essential role in vascular biology, modulating macrophage function and atherosclerosis progression. Recently, we have described the beneficial effect of combined activation of the ghrelin/GHS-R1a receptor and the scavenger receptor CD36 to induce macrophage cholesterol release through transcriptional activation of PPARγ. Although the interplay between CD36 and PPARγ in atherogenesis is well recognized, the contribution of the ghrelin receptor to regulate PPARγ remains unknown. Here, we demonstrate that ghrelin triggers PPARγ activation through a concerted signaling cascade involving Erk1/2 and Akt kinases, resulting in enhanced expression of downstream effectors LXRα and ABC sterol transporters in human macrophages. These effects were associated with enhanced PPARγ phosphorylation independently of the inhibitory conserved serine-84. Src tyrosine kinase Fyn was identified as being recruited to GHS-R1a in response to ghrelin, but failure of activated Fyn to enhance PPARγ Ser-84 specific phosphorylation relied on the concomitant recruitment of docking protein Dok-1, which prevented optimal activation of the Erk1/2 pathway. Also, substitution of Ser-84 preserved the ghrelin-induced PPARγ activity and responsiveness to Src inhibition, supporting a mechanism independent of Ser-84 in PPARγ response to ghrelin. Consistent with this, we found that ghrelin promoted the PI3-K/Akt pathway in a Gαq-dependent manner, resulting in Akt recruitment to PPARγ, enhanced PPARγ phosphorylation and activation independently of Ser-84, and increased expression of LXRα and ABCA1/G1. Collectively, these results illustrate a complex interplay involving Fyn/Dok-1/Erk and Gαq/PI3-K/Akt pathways to transduce in a concerted manner responsiveness of PPARγ to ghrelin in macrophages

Defining severe asthma - an approach to find new therapies

Asthma is a chronic inflammatory disease that has reached epidemic proportions worldwide. It is treatable in the majority of patients, but there is no cure. Moreover, a proportion of patients suffer from severe, difficult-to-control disease with daily symptoms and high morbidity, making it imperative that we continue to improve our understanding of the underlying mechanisms of this disease. Severe asthma is a heterogeneous condition. A systematic approach to identify specific asthma phenotypes, including clinical characteristics and inflammatory processes, is the first step toward individualized, logical therapy. This review focuses on the need to characterize severe asthma phenotypes and on novel, targeted molecular treatment options currently under development

Ghrelin exerts a proliferative effect on a rat pituitary somatotroph cell line via the mitogen-activated protein kinase pathway.

OBJECTIVES: Ghrelin is a brain-gut peptide with GH-releasing and appetite-inducing activities and a widespread tissue distribution. Ghrelin is the endogenous ligand of the GH secretagogue receptor type 1a (GHS-R1a), and both ghrelin and the GHS-R1a are expressed in the pituitary. There are conflicting data regarding the effects of ghrelin on cell proliferation. A positive effect on proliferation and activation of the mitogen-activated protein kinase (MAPK) pathway has been found in hepatoma, adipose, cardiomyocyte and prostate cell lines. However, ghrelin has also been shown to have anti-proliferative effects on breast, lung and thyroid cell lines. We therefore examined the effect of ghrelin on the rat pituitary cell line GH3. METHODS: RT-PCR was used for the detection of GHS-R1a and pre-proghrelin mRNA expression in GH3 cells. The effect of ghrelin on cell proliferation was studied using [(3)H]thymidine incorporation; cell counting and the activation of the MAPK pathway were studied using immunoblotting and inhibitors of the extracellular signal-regulated kinase 1 and 2 (ERK 1/2), protein kinase C (PKC) and tyrosine phosphatase pathways. RESULTS: GHS-R1a and ghrelin mRNA expression were detected in GH3 cells. Ghrelin, at 10(-10) to 10(-6) M concentrations, significantly increased [(3)H]thymidine incorporation (at 10(-9) M, 183+/-13% (means+/-s.e.m.) compared with untreated controls), while 12-phorbol 13-myristate acetate (PMA) at 10(-7) M (used as a positive control) caused a 212+/-14% increase. A reproducible stimulatory effect of desoctanoyl ghrelin was also observed on [(3)H]thymidine incorporation (135+/-5%; P<0.01 at 10(-9) M compared with control), as well as on the cell count (control 6.8 x 10(4)+/-8.7 x 10(3) cells/ml vs desoctanoyl ghrelin (10(-9) M) 1.04 x 10(5)+/-7.5 x 10(3) cells/ml; P<0.01). Ghrelin caused a significant increase in phosphorylated ERK 1/2 in immunoblotting, while desoctanoyl ghrelin showed a smaller but also significant stimulatory effect. The positive effect of ghrelin and desoctanoyl ghrelin on [(3)H]thymidine incorporation was abolished by the MAPK kinase inhibitor U0126, the PKC inhibitor GF109203X and the tyrosine kinase inhibitor tyrphostin 23, suggesting that the ghrelin-induced cell proliferation of GH3 cells is mediated both via a PKC-MAPK-dependent pathway and via a tyrosine kinase-dependent pathway. This could also be clearly demonstrated by Western blot analysis, where a transient increase in ERK 1/2 phosphorylation by ghrelin was attenuated by all three inhibitors. CONCLUSION: We have shown a novel role for ghrelin in stimulating the proliferation of a somatotroph pituitary tumour cell line, suggesting that ERK activation is involved in mediating the effects of ghrelin on cell proliferation. Desoctanoyl ghrelin showed a similar effect. As ghrelin has been shown to be expressed in both normal and adenomatous pituitary tissue, locally produced ghrelin may play a role in pituitary tumorigenesis via an autocrine/paracrine pathway

Novel molecular aspects of pituitary adenomas.

Ghrelin stimulates while somatostatin inhibits GH release and they thus serve as functional antagonists. We have compared their effects on cell proliferation. Ghrelin stimulates while somatostatin inhibits cell proliferation in most tissues and cell lines. Here we show that ghrelin and desoctanoyl ghrelin stimulate cell proliferation in rat pituitary cell line (GH3), and these effects could be inhibited with mitogen-activated protein kinase (MAPK), tyrosine kinase and protein kinase C inhibitors. Somatostatin and its analogs negatively regulate the growth of pituitary cells, and we now show that they inhibit MAPK activation. We hypothesised that one of the mechanisms involved in the somatostatin effect is a stimulation of cell cycle inhibitor p27, as pituitary adenomas have decreased p27 peptide content. Both octreotide and a new somatostatin analog SOM230 treatment resulted in an upregulation of p27 protein levels in human somatotrophinoma cells. In summary, we suggest that ghrelin and somatostatin have opposite effects on somatotroph cells not just at the level of GH release but also in terms of cell proliferation. Ghrelin may play a role in pituitary tumorigenesis via an autocrine/paracrine pathway. Our results also suggest that the antiproliferative effect of somatostatin analogs octreotide and SOM230 involve the up-regulation of p27 and down-regulation of the MAPK pathway in human somatotrophinomas

PPAR-gamma expression in pituitary tumours and the functional activity of the glitazones: evidence that any anti-proliferative effect of the glitazones is independent of the PPAR-gamma receptor.

OBJECTIVE: It has been reported that both normal pituitary and pituitary tumours express PPAR-gamma, a nuclear hormone receptor, the expression being more abundant in pituitary tumours, and that this is the basis for the reported antiproliferative effects of the thiazolidinedione, rosiglitazone, in animal models. However, the mechanisms for the responsivity to rosiglitazone have remained unclear. DESIGN AND MEASUREMENTS: To investigate this further, 'real-time' PCR was used to assess PPAR-gamma mRNA expression, and Western blotting and immunohistochemistry to study its protein expression, in 46 human pituitary tumours and normal pituitary tissue. Cell proliferation of the GH3 pituitary cell line was assessed by [3H]-thymidine-incorporation after 48 h rosiglitazone and pioglitazone (10(-4) M- 10(-10) M) treatment alone, or rosiglitazone in combination with the PPAR-gamma antagonist GW9662. RESULTS: PPAR-gamma mRNA and protein was found to be expressed in normal pituitary and was variably expressed in pituitary tumours, but were increased specifically in nonfunctioning pituitary adenomas. However, very little staining was observed with immunohistochemistry, with only occasional cell nuclei stained, and no difference was detectable between controls and tumours. Rosiglitazone at 10(-4) M and 10(-5) M concentrations inhibited cell proliferation (10(-4) M 14.0% +/- 1.5% and 10(-5) M 67% +/- 4%[mean +/- SEM]vs Control 100% +/- 3%, P < 0.0001) while lower concentrations showed no significant effect. Following withdrawal of rosiglitazone 10(-5) M, the cells fully recovered at a further 48 h, while lower doses showed a 'rebound' of stimulation. Pioglitazone was of similar potency to rosiglitazone in inhibiting proliferation. The PPAR-gamma antagonist did not show a significant reversal of the antiproliferative effect of rosiglitazone, and indeed suppressed proliferation on its own. CONCLUSIONS: Our data suggest that the antiproliferative action of rosiglitazone is probably not via PPAR-gamma

Enhanced protein kinase B/Akt signalling in pituitary tumours.

Pituitary tumours have previously been shown to harbour several abnormalities that cause deregulation of the cell cycle, particularly down-regulation of expression of the cyclin-dependent kinase inhibitor p27. However, it has been unclear whether these are the primary initiating events, or are secondary to other more proximate alterations in signalling pathways. In other cellular systems the Akt signalling pathway has been associated with downstream modulation of cell-cycle control. The aim of the present study was to test the hypothesis that Akt signalling is enhanced in pituitary tumours, and to see if changes in Akt expression are related to previous findings on low expression levels of the nuclear cell-cycle inhibitor p27 in pituitary tumours. We examined normal and adenomatous human pituitary tissue for mRNA and protein expression of Akt1, Akt2 and p27, and the activation of Akt, as well the phosphatase involved in the inactivation of Akt, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). In pituitary adenomas Akt1 and Akt2 mRNA were found to be over-expressed compared with normal pituitary, while PTEN transcripts showed similar levels between the two tissue types. Immunohistochemical expression of phospho-Akt was found to be higher in the tumours than normal pituitaries, while the protein expression of nuclear p27 and PTEN was lower in the adenomas. However, the expression of p27 and Akt were not directly correlated. PTEN sequencing revealed no mutation in the coding region of the gene in pituitary adenomas, and thus we did not locate a cause for the increased phosphorylation of Akt. In summary, we have shown over-expression and activation of the Akt pathway in pituitary tumours, and we speculate that cell-cycle changes observed in such tumours are secondary to these more proximate alterations. Since Akt is a major downstream signalling molecule of growth factor-liganded tyrosine kinase receptors, our data are most compatible with an abnormality at this level as the primary driver of pituitary tumorigenesis

Somatostatin analogues stimulate p27 expression and inhibit the MAP kinase pathway in pituitary tumours.

OBJECTIVES: Somatostatin (SST) analogues play an important role in the medical management of somatotroph pituitary adenomas and new agonists have the potential to be effective in a wider group of pituitary and other tumours. The anti-proliferative effect of SST occurs through multiple mechanisms, one of which is cell-cycle arrest, where p27, a cyclin-dependent kinase inhibitor, is an important regulator. We hypothesised that SST may upregulate p27 protein levels and downregulate the MAP kinase pathway in these tumours. METHODS: Human pituitary adenoma cells and rat pituitary cell line (GH3) were cultured and treated in vitro with octreotide and the broad-spectrum SST agonist SOM230 (pasireotide). Immunoblotting for p27 and phospho-ERK (pERK) was performed and proliferation assessed by [3H]-thymidine incorporation. Histological samples from acromegalic patients treated with octreotide before surgery were immunostained for p27 and compared to samples from untreated patients matched for sex, age, tumour size, extension and invasiveness. RESULTS: We detected upregulation of p27 protein levels with SST analogue treatment in vitro in human pituitary adenoma samples. pERK1/2 was inhibited by SST analogues in both the human samples and GH3 cells. SST and its analogues inhibited the proliferation of GH3 cells. p27 immunostaining was stronger in samples from patients with longer preoperative octreotide treatment (more than 6 months) than in samples from patients with shorter treatment periods. CONCLUSIONS: This study demonstrates that SST-mediated growth inhibition is associated with the downregulation of pERK and upregulation of p27. More potent and broader-spectrum SST analogues are likely to play an increasing role in the treatment of tumours, where the MAP kinase pathway is overactivated