Dual regulation of ß2-adrenoceptor messenger RNA expression in human lung fibroblasts by ß2-cAMP signaling; delayed upregulated inhibitors oppose a rapid in onset, direct stimulation of gene expression.

Based on their bronchodilatory effect, ß2-adrenoceptor agonists constitute essential elements in the treatment of bronchial asthma and COPD. As treatment with ß2-adrenoceptor agonists has been associated with worsening of airway hyper-reactivity, possibly because of loss of ß-adrenoceptor function, molecular mechanism of the regulation of ß2-adrenoceptor expression were studied. MRC-5 human lung fibroblasts were cultured in absence or presence of test substances followed by ß2-adrenoceptor messenger RNA (mRNA) determination by qPCR. After inhibition of mRNA synthesis by actinomycin D, ß2-adrenoceptor mRNA decreased with a half-life of 23 min, whereas inhibition of protein synthesis by cycloheximide caused an about 5- and 6-fold increase within 1.5 and 4 h, respectively. ß2-Adrenoceptor mRNA was increased by about 100 % after 1 h exposure to formoterol or olodaterol but decreased by about 60 % after 4 h agonist exposure. Both effects of ß2-adrenoceptor agonists were mimicked by forskolin, a direct activator of adenylyl cyclase and cholera toxin, which stimulates adenylyl cyclase by permanent activation of Gs. ß2-Adrenoceptor agonist-induced upregulation of ß2-adrenoceptor mRNA was blocked by the ß2-adrenoceptor antagonist ICI 118551 and prevented by actinomycin D, but not by cycloheximide. Moreover, in presence of cycloheximide, ß2-adrenoceptor agonist-induced reduction in ß2-adrenoceptor mRNA was converted into stimulation, resulting in a more than 10-fold increase. In conclusion, expression of ß2-adrenoceptors in human lung fibroblasts is highly regulated at transcriptional level. The ß2-adrenoceptor gene is under strong inhibitory control of short-living suppressor proteins. ß2-Adrenoceptor activation induces via adenylyl cyclase - cyclic adenosine monophosphate (cAMP) signaling a rapid in onset direct stimulation of the ß2-adrenoceptor gene transcription, an effect opposed by a delayed upregulation of inhibitory factors.

Pro-fibrotic processes in human lung fibroblasts are driven by an autocrine/paracrine endothelinergic system.

BACKGROUND AND PURPOSE: Since endothelin (ET) may act as pro-fibrotic mediator, expression and release of ET isoforms, their receptors and potential pro-fibrotic ET effects were studied in human lung fibroblasts. EXPERIMENTAL APPROACH: MRC-5 and primary human lung fibroblasts (phLFb) were cultured. Expression of prepro-ET isoforms was determined by qPCR and release of ET-1 by elisa. ET receptor function was analysed by real-time measurement of dynamic mass redistribution (DMR). Incorporation of [(3) H]-thymidine was determined as measure of proliferation and that of [(3) H]-proline for collagen synthesis. Phospho-p42/44 MAP kinase was determined by Western blot. KEY RESULTS: ET-1 is the predominant ET in human lung fibroblasts (hLF), and TGF-ß caused a further, selective and sustained up-regulation of ET-1 resulting in increased extracellular ET-1 accumulation. hLFb express mRNA encoding ET-A and ET-B receptors. Expression of both receptors was confirmed at protein level. ET-1 induced marked DMR signals, an effect that involved ET-A and ET-B receptors. Stimulatory effects of ET-1 on hLFb proliferation and collagen synthesis were mediated exclusively via ET-A receptors. ET-1, again via ET-A receptors, induced rapid activation of ERK MAPK, shown to be a crucial cellular signal in ET-1-induced collagen synthesis. ET-1-induced activation of ERK and collagen synthesis was, in contrast to corresponding effect of a muscarinic agonist, largely insensitive to pertussis toxin. CONCLUSIONS AND IMPLICATIONS: hLFb are endowed with all elements necessary to build a functional autocrine/paracrine endothelinergic system, which appears to drive pro-fibrotic airway and lung remodelling processes, effects for which only ET-A, but not ET-B receptors appear to be of significance.

Characterization of proliferative effects of insulin, insulin analogues and insulin-like growth factor-1 (IGF-1) in human lung fibroblasts.

Insulin has been approved for inhaled application, but safety concerns remain, because of un-physiologically high insulin concentrations in the lung. Since insulin may act as growth factor, possible proliferative effects of insulin, insulin analogues and insulin-like growth factor-1 (IGF-1) on human lung fibroblasts were studied. As measure of proliferation [(3)H]-thymidine incorporation was studied in HEL-299, MRC-5, IMR-90 and primary human lung fibroblasts. In all cells, mRNA encoding IGF-1 receptors and two variants of insulin receptors was detected. Insulin and IGF-1 stimulated [(3)H]-thymidine incorporation in all cells. Comparison of the concentration-dependent effects in HEL-299 cells showed that IGF-1 and insulin glargine were more potent (EC(50), 3 and 6 nM) and more effective (maximum increase, by 135-150%) than insulin and insulin detemir (EC(50), 22 and 110 nM; maximum increase: by 80%). Proliferative effects of IGF-1 and insulin were inhibited to the same extent by an antibody (1H7) directed against the IGF-1 receptor α-subunit. Insulin-induced stimulation of [(3)H]-thymidine incorporation was reduced by 83% after siRNA-mediated down-regulation of IGF-1 receptor by about 75%, but not affected by a similar down-regulation of the insulin receptor. Insulin and IGF-1 caused rapid up-regulation of the early genes FOS, EGR-1 and EGR-2 as well as of the gene coding for IGF-1. In conclusion, in human lung fibroblasts insulin exerts marked proliferative effects and the pharmacological profile of this response as well as specific receptor knock-down experiments suggest mediation via IGF-1 receptors. The risk of unwanted structural lung alterations by long-term inhalative application of insulin should be considered.

Species differences in expression pattern of arginase isoenzymes and differential effects of arginase inhibition on collagen synthesis in human and rat pulmonary fibroblasts.

Arginase was shown to be up-regulated in different animal models of inflammatory and fibrotic airway diseases. Since arginase provides L-ornithine, one precursor for L-proline, an essential substrate for collagen synthesis, it has been suggested that arginase might be a key enzyme in airway remodelling. The present study aimed to characterize expression of arginase isoenzymes in rat and human pulmonary fibroblasts, and to test whether arginase inhibition affects collagen synthesis. In primary rat tracheal and lung fibroblasts, mRNA for arginase I and II could be detected, with arginase I as predominant isoenzyme. In contrast, in human lung fibroblasts (primary cells and different cells lines) mRNA levels for arginase I were at or below detection limit whereas arginase II mRNA was markedly higher than in rat pulmonary fibroblasts. Arginase activity in rat tracheal and lung fibroblasts was between 20 and 30 mU/mg protein, but was below detection limit (2.5 mU/mg) in human lung fibroblasts. In rat tracheal and lung fibroblasts cultured in proline-free medium, arginase inhibition by N(omega)-hydroxy-nor-L-arginine caused a reduction by about one-third of basal collagen I accumulation (determined by western blot analysis) and largely attenuated transforming growth factor beta 1 (TGF-beta(1))-induced increase in collagen accumulation, whereas basal and TGF-beta(1)-induced collagen accumulation by human lung fibroblasts was not affected by arginase inhibition. In conclusion, arginase isoenzymes reveal a species specific expression pattern. Arginase contributes significantly to L-proline supply for collagen synthesis in rat fibroblasts, in which arginase I is the predominant isoenzyme, but not in human fibroblasts, in which arginase II is the only isoenzyme expressed.

Role of Epac1 in mediating anti-proliferative effects of prostanoid EP(2) receptors and cAMP in human lung fibroblasts.

In lung fibroblasts, proliferation is inhibited by activation of EP(2) prostanoid receptors which are known to couple to adenylyl cyclase. Beside the classic target of cAMP, protein kinase A (PKA), alternative cAMP effectors have been identified, among them Epac (exchange protein activated by cAMP). The present study aimed to illuminate transduction pathways mediating the anti-proliferative effects of EP(2) receptors in lung fibroblasts. Proliferative activity of human lung fibroblasts was determined by measuring [(3)H]-thymidine incorporation. The selective EP(2) receptor agonist butaprost inhibited [(3)H]-thymidine incorporation by 75%, an effect mimicked by forskolin, the phosphodiesterase inhibitor IBMX, the stable cAMP analogues dibutyryl-cAMP and bromo-cAMP, as well as by the Epac selective cAMP analogues 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, whereas the PKA selective agonist 6-Bnz-cAMP was inactive. The PKA inhibitor Rp-8-Br-cAMPS inhibited butaprost-induced phosphorylation of CREB (cAMP response element-binding protein), but did not affect butaprost-induced inhibition of [(3)H]-thymidine incorporation. Partial knockdown of Epac1 by specific siRNA transfection resulted in a marked attenuation of the inhibitory potency of butaprost, whereas transfection of Epac2 siRNA or non-silencing siRNA did not affect the effectiveness of butaprost to inhibit [(3)H]-thymidine incorporation. In conclusion, Epac1 rather than the classic cAMP effector PKA is a crucial element in the signal transduction pathway mediating anti-proliferative effects of EP(2) receptor activation.

Methicillin-resistant Staphylococcus aureus in human milk.

We collected and analyzed 500 samples of human milk, from five Brazilian cities (100 from each) to detect methicillin-resistant strains of Staphylococcus aureus (MRSA) producing enterotoxins. We found 57 strains of MRSA, and the mecA gene, responsible for resistance, was detected in all of them using a specific molecular probe. We examined 40 strains for the presence of four enterotoxins, after selecting a subset that included all strains from each region, except for the largest sample, from which 10 were randomly selected. Among these two presented enterotoxin B, and growth in human colostrum and trypicase soy broth. After 5 h of incubation at 37 degrees C, population sizes were already higher than 9.4 x 10(5) UFC/ml and enterotoxin was released into culture medium and colostrum. Our results stress the importance of hygiene, sanitary measures, and appropriate preservation conditions to avoid the proliferation of S. aureus in human milk.

Methicillin-resistant Staphylococcus aureus in human milk

We collected and analyzed 500 samples of human milk, from five Brazilian cities (100 from each) to detect methicillin-resistant strains of Staphylococcus aureus (MRSA) producing enterotoxins. We found 57 strains of MRSA, and the mecA gene, responsible for resistance, was detected in all of them using a specific molecular probe. We examined 40 strains for the presence of four enterotoxins, after selecting a subset that included all strains from each region, except for the largest sample, from which 10 were randomly selected. Among these two presented enterotoxin B, and growth in human colostrum and trypicase soy broth. After 5 h of incubation at 37§C, population sizes were already higher than 9.4 x 105 UFC/ml and enterotoxin was released into culture medium and colostrum. Our results stress the importance of hygiene, sanitary measures, and appropriate preservation conditions to avoid the proliferation of S. aureus in human milk.