Differential effects of RGS proteins on Gαq and Gα11 activity

Heterotrimeric G proteins play a pivotal role in GPCR signalling; they link receptors to intracellular effectors and their inactivation by RGS proteins is a key factor in resetting the pathway following stimulation. The precise GPCR:G protein:RGS combination determines the nature and duration of the response. Investigating the activity of particular combinations is difficult in cells which contain multiples of each component. We have therefore utilised a previously characterised yeast system to express mammalian proteins in isolation. Human Gαq and Gα11 spontaneously activated the yeast pheromone-response pathway by a mechanism which required the formation of Gα-GTP. This provided an assay for the specific activity of human RGS proteins. RGS1, RGS2, RGS3 and RGS4 inhibited the spontaneous activity of both Gαq and Gα11 but, in contrast, RGS5 and RGS16 were much less effective against Gα11 than Gαq. Interestingly, RGS2 and RGS3 were able to inhibit signalling from the constitutively active Gαq QL/Gα11 QL mutants, confirming the GAP-independent activity of these RGS proteins. To determine if the RGS-Gα specificity was maintained under conditions of GPCR stimulation, minor modifications to the C-terminus of Gαq/Gα11 enabled coupling to an endogenous receptor. RGS2 and RGS3 were effective inhibitors of both Gα subunits even at high levels of receptor stimulation, emphasising their GAP-independent activity. At low levels of stimulation RGS5 and RGS16 retained their differential Gα activity, further highlighting that RGS proteins can discriminate between two very closely related Gα subunits

Clean & Fast Amide Couplings in Water

EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) is a carbodiimide that is well known reagent for the amide coupling reactions between an amine and carboxylic acid in the presence of a base. It is the commonly used coupling agent in industries because it produces a water-soluble urea byproduct, which can be easily removed by an aqueous work-up. However, our methodology, the desired amide product precipitates out of the reaction mixture, and no additional work-up is required.3 Using aqueous micelles of PS-750-M as a solvent dramatically increases the rate of reaction.2 The filtrate recovered can be recycled to perform several coupling runs after post-reaction manipulation

Suppression of autophagy by FIP200 deletion leads to osteopenia in mice through the inhibition of osteoblast terminal differentiation

Autophagy is a conserved lysosomal degradation process that has important roles in both normal human physiology and disease. However, the function of autophagy in bone homeostasis is not well understood. Here, we report that autophagy is activated during osteoblast differentiation. Ablation of focal adhesion kinase family interacting protein of 200 kD (FIP200), an essential component of mammalian autophagy, led to multiple autophagic defects in osteoblasts including aberrantly increased p62 expression, deficient LC3‐II conversion, defective autophagy flux, absence of GFP‐LC3 puncta in FIP200‐null osteoblasts expressing transgenic GFP‐LC3, and absence of autophagosome‐like structures by electron microscope examination. Osteoblast‐specific deletion of FIP200 led to osteopenia in mice. Histomorphometric analysis revealed that the osteopenia was the result of cell‐autonomous effects of FIP200 deletion on osteoblasts. FIP200 deletion led to defective osteoblast terminal differentiation in both primary bone marrow and calvarial osteoblasts in vitro. Interestingly, both proliferation and differentiation were not adversely affected by FIP200 deletion in early cultures. However, FIP200 deletion led to defective osteoblast nodule formation after initial proliferation and differentiation. Furthermore, treatment with autophagy inhibitors recapitulated the effects of FIP200 deletion on osteoblast differentiation. Taken together, these data identify FIP200 as an important regulator of bone development and reveal a novel role of autophagy in osteoblast function through its positive role in supporting osteoblast nodule formation and differentiation. © 2013 American Society for Bone and Mineral Research.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/100319/1/jbmr1971.pd

Thyroid and pituitary gland development from hatching through metamorphosis of a teleost flatfish, the Atlantic halibut

Fish larval development, not least the spectacular process of flatfish metamorphosis, appears to be under complex endocrine control, many aspects of which are still not fully elucidated. In order to obtain data on the functional development of two major endocrine glands, the pituitary and the thyroid, during flatfish metamorphosis, histology, immunohistochemistry and in situ hybridization techniques were applied on larvae of the Atlantic halibut (Hippoglossus hippoglossus), a large, marine flatfish species, from hatching through metamorphosis. The material was obtained from a commercial hatchery. Larval age is defined as day-degrees (D =accumulated daily temperature from hatching). Sporadic thyroid follicles are first detected in larvae at 142 D (27 days post-hatch), prior to the completion of yolk sack absorption. Both the number and activity of the follicles increase markedly after yolk sack absorption and continue to do so during subsequent development. The larval triiodothyronine (T3) and thyroxine (T4) content increases, subsequent to yolk absorption, and coincides with the proliferation of thyroid follicles. A second increase of both T3 and T4 occurs around the start of metamorphosis and the T3 content further increases at the metamorphic climax. Overall, the T3 content is lower than T4. The pituitary gland can first be distinguished as a separate organ at the yolk sack stage. During subsequent development, the gland becomes more elongated and differentiates into neurohypophysis (NH), pars distalis (PD) and pars intermedia (PI). The first sporadic endocrine pituitary cells are observed at the yolk sack stage, somatotrophs (growth hormone producing cells) and somatolactotrophs (somatolactin producing cells) are first observed at 121 D (23 days post-hatch), and lactotrophs (prolactin producing cells) at 134 D (25 days post-hatch). Scarce thyrotrophs are evident after detection of the first thyroid follicles (142 D ), but coincident with a phase in which follicle number and activity increase (260 D ). The somatotrophs are clustered in the medium ventral region of the PD, lactotrophs in the anterior part of the PD and somatolactotrophs are scattered in the mid and posterior region of the pituitary. At around 600 D , coinciding with the start of metamorphosis, somatolactotrophs are restricted to the interdigitating tissue of the NH. During larval development, the pituitary endocrine cells become more numerous. The present data on thyroid development support the notion that thyroid hormones may play a significant role in Atlantic halibut metamorphosis. The time of appearance and the subsequent proliferation of pituitary somatotrophs, lactotrophs, somatolactotrophs and thyrotrophs indicate at which stages of larval development and metamorphosis these endocrine cells may start to play active regulatory roles.This work has been carried out within the projects ‘‘Endocrine Control as a Determinant of Larval Quality in Fish Aquaculture’’ (CT-96-1422) and ‘‘Arrested development: The Molecular and Endocrine Basis of Flatfish Metamorphosis’’ (Q5RS-2002-01192), with financial support from the Commission of the European Communities. However, it does not necessarily reflect the Commission’s views and in no way anticipates its future policy in this area. This project was further supported by the Swedish Council for Agricultural and Forestry Research and Pluriannual funding to CCMAR by the Portuguese Science and Technology Council

PYK2 selectively mediates signals for growth versus differentiation in response to stretch of spontaneously active vascular smooth muscle.

Stretch of vascular smooth muscle stimulates growth and proliferation as well as contraction and expression of contractile/cytoskeletal proteins, all of which are also regulated by calcium-dependent signals. We studied the role of the calcium- and integrin-activated proline-rich tyrosine kinase 2 (PYK2) in stretch-induced responses of the rat portal vein loaded by a hanging weight ex vivo. PYK2 phosphorylation at Tyr-402 was increased both by a 10-min stretch and by organ culture with load over several days. Protein and DNA synthesis were reduced by the novel PYK2 inhibitor PF-4594755 (0.5-1 μmol/L), while still sensitive to stretch. In 3-day organ culture, PF-4594755 caused maintained myogenic spontaneous activity but did not affect contraction in response to high-K(+) (60 mmol/L) or to α1-adrenergic stimulation by cirazoline. Basal and stretch-induced PYK2 phosphorylation in culture were inhibited by PF-4594755, closely mimicking inhibition of non-voltage-dependent calcium influx by 2-APB (30 μmol/L). In contrast, the L-type calcium channel blocker, nifedipine (1 μmol/L) eliminated stretch-induced but not basal PYK2 phosphorylation. Stretch-induced Akt and ERK1/2 phosphorylation was eliminated by PF-4594755. PYK2 inhibition had no effect on mRNA expression of several smooth muscle markers, and stretch-sensitive SM22α synthesis was preserved. Culture of portal vein with the Ang II inhibitor losartan (1 μmol/L) eliminated stretch sensitivity of PYK2 and Akt phosphorylation, but did not affect mRNA expression of smooth muscle markers. The results suggest that PYK2 signaling functionally distinguishes effects of voltage- and non-voltage-dependent calcium influx. A small-molecule inhibitor of PYK2 reduces growth and DNA synthesis but does not affect contractile differentiation of vascular smooth muscle

Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

Signalling pathways that control endothelial cell (EC) permeability, leukocyte adhesion and inflammation are pivotal for atherosclerosis initiation and progression. Here we demonstrate that the Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), which has been implicated in inflammation, is abundantly expressed in ECs and in atherosclerotic plaques from mice and humans. On the basis of endothelial-specific MAP4K4 gene silencing and gene ablation experiments in Apoe(-/-) mice, we show that MAP4K4 in ECs markedly promotes Western diet-induced aortic macrophage accumulation and atherosclerotic plaque development. Treatment of Apoe(-/-) and Ldlr(-/-) mice with a selective small-molecule MAP4K4 inhibitor also markedly reduces atherosclerotic lesion area. MAP4K4 silencing in cultured ECs attenuates cell surface adhesion molecule expression while reducing nuclear localization and activity of NFkappaB, which is critical for promoting EC activation and atherosclerosis. Taken together, these results reveal that MAP4K4 is a key signalling node that promotes immune cell recruitment in atherosclerosis

Effect of Stress on Viral–Bacterial Synergy in Bovine Respiratory Disease: Novel Mechanisms to Regulate Inflammation

The severity of bovine respiratory infections has been linked to a variety of factors, including environmental and nutritional changes, transportation, and social reorganization of weaned calves. Fatal respiratory infections, however, usually occur when a primary viral infection compromises host defences and enhances the severity of a secondary bacterial infection. This viral–bacterial synergy can occur by a number of different mechanisms and disease challenge models have been developed to analyse host responses during these respiratory infections. A primary bovine herpesvirus-1 (BHV-1) respiratory infection followed by a secondary challenge with Mannheimia haemolytica results in fatal bovine respiratory disease (BRD) and host responses to these two pathogens have been studied extensively. We used this disease model to demonstrate that stress significantly altered the viral–bacterial synergy resulting in fatal BRD. Functional genomic analysis revealed that BHV-1 infection enhanced toll-like receptors (TLR) expression and increased pro-inflammatory responses which contribute to the severity of a Mannheimia haemolytica infection. TLRs play a critical role in detecting bacterial infections and inducing pro-inflammatory responses. It is difficult to understand, however, how stress-induced corticosteroids could enhance this form of viral–bacterial synergy. Nuclear translocation of the glucocorticoid receptor activates cell signalling pathways which inhibit both TLR signalling and pro-inflammatory responses. The apparent conundrum between stress-induced corticosteroids and enhanced BRD susceptibility is discussed in terms of present data and previous investigations of stress and respiratory disease

The retinoid anticancer signal: mechanisms of target gene regulation

Retinoids induce growth arrest, differentiation, and cell death in many cancer cell types. One factor determining the sensitivity or resistance to the retinoid anticancer signal is the transcriptional response of retinoid-regulated target genes in cancer cells. We used cDNA microarray to identify 31 retinoid-regulated target genes shared by two retinoid-sensitive neuroblastoma cell lines, and then sought to determine the relevance of the target gene responses to the retinoid anticancer signal. The pattern of retinoid responsiveness for six of 13 target genes (RARβ2, CYP26A1, CRBP1, RGS16, DUSP6, EGR1) correlated with phenotypic retinoid sensitivity, across a panel of retinoid-sensitive or -resistant lung and breast cancer cell lines. Retinoid treatment of MYCN transgenic mice bearing neuroblastoma altered the expression of five of nine target genes examined (RARβ2, CYP26A1, CRBP1, DUSP6, PLAT) in neuroblastoma tumour tissue in vivo. In retinoid-sensitive neuroblastoma, lung and breast cancer cell lines, direct inhibition of retinoid-induced RARβ2 expression blocked induction of only one of eight retinoid target genes (CYP26A1). DNA demethylation, histone acetylation, and exogenous overexpression of RARβ2 partially restored retinoid-responsive CYP26A1 expression in RA-resistant MDA-MB-231 breast, but not SK-MES-1 lung, cancer cells. Combined, rather than individual, inhibition of DUSP6 and RGS16 was required to block retinoid-induced growth inhibition in neuroblastoma cells, through phosphorylation of extracellular-signal-regulated kinase. In conclusion, sensitivity to the retinoid anticancer signal is determined in part by the transcriptional response of key retinoid-regulated target genes, such as RARβ2, DUSP6, and RGS16

Skp2B Overexpression Alters a Prohibitin-p53 Axis and the Transcription of PAPP-A, the Protease of Insulin-Like Growth Factor Binding Protein 4

We previously reported that the degradation of prohibitin by the SCF(Skp2B) ubiquitin ligase results in a defect in the activity of p53. We also reported that MMTV-Skp2B transgenic mice develop mammary gland tumors that are characterized by an increased proteolytic cleavage of the insulin-like growth factor binding protein 4 (IGFBP-4), an inhibitor of IGF signaling. However, whether a link exists between a defect in p53 activity and proteolysis of IGFBP-4 was not established.We analyzed the levels of pregnancy-associated plasma protein A (PAPP-A), the protease of IGFBP-4, in MMTV-Skp2B transgenic mice and found that PAPP-A levels are elevated. Further, we found a p53 binding site in intron 1 of the PAPP-A gene and that both wild type and mutant p53 bind to this site. However, binding of wild type p53 results in the transcriptional repression of PAPP-A, while binding of mutant p53 results in the transcriptional activation of PAPP-A. Since MMTV-Skp2B mice express wild type p53 and yet show elevated levels of PAPP-A, at first, these observations appeared contradictory. However, further analysis revealed that the defect in p53 activity in Skp2B overexpressing cells does not only abolish the activity of wild type of p53 but actually mimics that of mutant p53. Our results suggest that in absence of prohibitin, the half-life of p53 is increased and like mutant p53, the conformation of p53 is denatured.These observations revealed a novel function of prohibitin as a chaperone of p53. Further, they suggest that binding of denatured p53 in intron 1 causes an enhancer effect and increases the transcription of PAPP-A. Therefore, these findings indicate that the defect in p53 function and the increased proteolysis of IGFBP-4, we had observed, represent two components of the same pathway, which contributes to the oncogenic function of Skp2B