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

Transcriptomal profiling of the cellular response to DNA damage mediated by Slug (Snai2)

Snai2-deficient cells are radiosensitive to DNA damage. The function of Snai2 in response to DNA damage seems to be critical for its function in normal development and cancer. Here, we applied a functional genomics approach that combined gene-expression profiling and computational molecular network analysis to obtain global dissection of the Snai2-dependent transcriptional response to DNA damage in primary mouse embryonic fibroblasts (MEFs), which undergo p53-dependent growth arrest in response to DNA damage. Although examination of the response showed that overall expression of p53 target gene expression patterns was similarly altered in both control and Snai2-deficient cells, we have identified and validated candidate Snai2 target genes linked to Snai2 gene function in response to DNA damage. This work defines for the first time the effect of Snai2 on p53 target genes in cells undergoing growth arrest, elucidates the Snai2-dependent molecular network induced by DNA damage, points to novel putative Snai2 targets, and suggest a mechanistic model, which has implications for cancer management

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

Non-Overlapping Functions for Pyk2 and FAK in Osteoblasts during Fluid Shear Stress-Induced Mechanotransduction

Mechanotransduction, the process by which cells convert external mechanical stimuli such as fluid shear stress (FSS) into biochemical changes, plays a critical role in maintenance of the skeleton. We have proposed that mechanical stimulation by FSS across the surfaces of bone cells results in formation of unique signaling complexes called mechanosomes that are launched from sites of adhesion with the extracellular matrix and with other bone cells [1]. Deformation of adhesion complexes at the cell membrane ultimately results in alteration of target gene expression. Recently, we reported that focal adhesion kinase (FAK) functions as a part of a mechanosome complex that is required for FSS-induced mechanotransduction in bone cells. This study extends this work to examine the role of a second member of the FAK family of non-receptor protein tyrosine kinases, proline-rich tyrosine kinase 2 (Pyk2), and determine its role during osteoblast mechanotransduction. We use osteoblasts harvested from mice as our model system in this study and compared the contributions of Pyk2 and FAK during FSS induced mechanotransduction in osteoblasts. We exposed Pyk2+/+ and Pyk2−/− primary calvarial osteoblasts to short period of oscillatory fluid flow and analyzed downstream activation of ERK1/2, and expression of c-fos, cyclooxygenase-2 and osteopontin. Unlike FAK, Pyk2 was not required for fluid flow-induced mechanotransduction as there was no significant difference in the response of Pyk2+/+ and Pyk2−/− osteoblasts to short periods of fluid flow (FF). In contrast, and as predicted, FAK−/− osteoblasts were unable to respond to FF. These data indicate that FAK and Pyk2 have distinct, non-redundant functions in launching mechanical signals during osteoblast mechanotransduction. Additionally, we compared two methods of generating FF in both cell types, oscillatory pump method and another orbital platform method. We determined that both methods of generating FF induced similar responses in both primary calvarial osteoblasts and immortalized calvarial osteoblasts

Abrogation of Cbl–PI3K Interaction Increases Bone Formation and Osteoblast Proliferation

Cbl is an adaptor protein and E3 ligase that plays both positive and negative roles in several signaling pathways that affect various cellular functions. Tyrosine 737 is unique to Cbl and phosphorylated by Src family kinases. Phosphorylated CblY737 creates a binding site for the p85 regulatory subunit of phosphatidylinositol 3 kinase (PI3K) that also plays an important role in the regulation of bone homeostasis. To investigate the role of Cbl–PI3K interaction in bone homeostasis, we examined knock-in mice in which the PI3K binding site on Cbl was ablated due to the substitution of tyrosine 737 to phenylalanine (CblYF/YF, YF mice). We previously reported that bone volume in these mice is increased due to decreased osteoclast function (Adapala et al., J Biol Chem 285:36745–36758, 19). Here, we report that YF mice also have increased bone formation and osteoblast numbers. In ex vivo cultures bone marrow-derived YF osteoblasts showed increased Col1A expression and their proliferation was also significantly augmented. Moreover, proliferation of MC3T3-E1 cells was increased after treatment with conditioned medium generated by culturing YF bone marrow stromal cells. Expression of stromal derived factor-1 (SDF-1) was increased in YF bone marrow stromal cells compared to wild type. Increased immunostaining of SDF-1 and CXCR4 was observed in YF bone marrow stromal cells compared to wild type. Treatment of YF condition medium with neutralizing anti-SDF-1 and anti-CXCR4 antibodies attenuated MC3T3-E1 cell proliferation. Cumulatively, these results show that abrogation of Cbl–PI3K interaction perturbs bone homeostasis, affecting both osteoclast function and osteoblast proliferation

Enhanced prediction of breast cancer prognosis by evaluating expression of p53 and prostate-specific antigen in combination

p53 gene mutation is the most common genetic alteration in neoplastic diseases, including breast cancer, for which p53 alteration may indicate poor prognosis. Recent clinical evidence suggests that prostate-specific antigen (PSA) expression may identify breast cancer patients with favourable outcome. Assessment of p53 and PSA in combination, potentially offering improved prediction, has not yet been performed. Extracts from 952 primary breast carcinomas were assayed for PSA and p53 by quantitative enzyme-linked immunosorbent assays (ELISAs) developed by the authors. Concentrations of each marker were classified as negative or positive on the basis of median and 30th percentile cut-off points for p53 and PSA respectively. Patients followed for a median of 6 years having different combinations of negative or positive status for PSA and p53 were compared with respect to the relative risks (RRs) for relapse and death by Cox proportional hazards regression analysis, in which an interaction term was also evaluated, and with respect to disease-free survival (DFS) and overall survival (OS) probabilities by Kaplan–Meier plots and log-rank tests. Multivariate models were adjusted for oestrogen and progesterone receptor status, nodal status, patient age, tumour size, DNA ploidy, S phase fraction and receipt of chemotherapy. Interactions were not found between the status of PSA and p53 in the Cox models, in which PSA-negativity (RR = 1.47, P = 0.020 for DFS, and RR = 1.49, P = 0.023 for OS) and p53-positivity (RR = 1.46, P = 0.017 for DFS, and RR = 1.41, P = 0.033 for OS) were individually associated with prognosis. Evaluation of a combined three-level variable revealed that PSA(–)/p53(+) patients had significantly higher risks for relapse (RR = 2.13, P < 0.001) and death (RR = 2.08, P = 0.001) than PSA(+)/p53(–) patients, and that patients positive or negative for both markers had intermediate risks for the outcome events in the same multivariate analysis (RR = 1.45 for both DFS and OS). The results of our study demonstrate that the assessment of combined PSA and p53 expression status by ELISAs, easily applicable to breast tumour extracts prepared for steroid hormone receptor analyses, may stratify breast cancer patients into groups differing by relapse and death risks of greater magnitude than offered by the assessment of either p53 or PSA alone. © 1999 Cancer Research Campaig