Urocortin stimulates the ERK1/2 signaling pathway and the proliferation of HeLa cells via CRF receptor 1.

Corticotropin-releasing factor (CRF) stimulates adrenocorticotropic hormone (ACTH) secretion from the pituitary gland and is an essential regulator of the hypothalamic-pituitary-adrenocortical axis. Isoforms of CRF receptor are known to mediate the effects of urocortin stress ligands on the regulation of stress responses, anxiety, and feeding behavior; however, urocortin stress ligands also influence cell proliferation. In view of the tumor-promoting capacity of prolonged stress, here we investigated (a) the effect of urocortin on cell proliferative signaling via extracellular signal-regulated kinase 1/2, (b) the expression and cellular distribution of the specific CRF receptor isoforms, and (c) the intracellular localization of phosphorylated ERK1/2 in HeLa cells. Stimulation of cell proliferation was observed in the presence of 10 nm urocortin. Our data also suggest that MAP kinase MEK, the transcription factors E2F-1 and p53, and PKB/Akt are involved in this process. These findings may have therapeutic relevance for the targeted treatment of various malignancies.

Prolonged treatment with the proteasome inhibitor MG-132 induces apoptosis in PC12 rat pheochromocytoma cells.

Rat pheochromocytoma (PC12) cells were treated with the proteasome inhibitor MG-132 and morphological changes were recorded. Initially, neuronal differentiation was induced but after 24 h signs of morphological deterioration became apparent. We performed nuclear staining, flow cytometry and WST-1 assay then analyzed signal transduction pathways involving Akt, p38 MAPK (Mitogen-Activated Protein Kinase), JNK (c-Jun N-terminal Kinase), c-Jun and caspase-3. Stress signaling via p38, JNK and c-Jun was active even after 24 h of MG-132 treatment, while the survival-mediating Akt phosphorylation declined and the executor of apoptosis (caspase-3) was activated by that time and apoptosis was also observable. We examined subcellular localization of stress signaling components, applied kinase inhibitors and dominant negative H-Ras mutant-expressing PC12 cells in order to decipher connections of stress-mediating pathways. Our results are suggestive of that treatment with the proteasome inhibitor MG-132 has a biphasic nature in PC12 cells. Initially, it induces neuronal differentiation but prolonged treatments lead to apoptosis.

Urocortin stimulates ERK1/2 phosphorylation and proliferation but reduces ATP production of MCF7 breast cancer cells.

Urocortins are members of the stress-related corticotropin-releasing factor family. Small amounts of them are present in the circulation and they are produced locally in various tissues of higher vertebrates. Aside from regulating circulation, or food uptake they also influence, via auto- and paracrine mechanisms, cell proliferation. In the present study we investigated in MCF7 human breast cancer cells the effect of urocortin onto mitogenic signaling via ERK1/2. Our results revealed that already 10 nM urocortin could stimulate the phosphorylation of these kinases and cell proliferation of MCF7 cells while ATP production was reduced when kept in the presence of the peptide up to two days. We examined the expression and contribution of the specific receptors of urocortin to the activation of ERK1/2 and to cell proliferation, the intracellular distribution of phosphorylated ERK1/2, and the involvement of additional proteins like PKA, PKB/Akt, MEK, p53, Rb and E2F-1 behind the observed phenomena.

FTY720 (Gilenya) treatment prevents spontaneous autoimmune myocarditis and dilated cardiomyopathy in transgenic HLA-DQ8-BALB/c mice.

Although dilated cardiomyopathy (DCM) is often caused by viral infections, it frequently involves autoimmune mechanisms associated with particular HLA-DR and DQ alleles. Our homozygous HLA-DQ8Ab(0) transgenic mice in the BALB/c background (HLA-DQ8(BALB/c)-Tg) developed early and progressive fatal heart failure from 4 to 5 weeks of age. Clinical signs of the disease included cyanotic eyes, tachycardia with dyspnea (from pale to cyanotic limbs), and terminal whole body edema. Sick mice had extremely dilated hearts, enlarged liver and spleen, and pleural/peritoneal effusion. Histology of the heart showed extensive heart muscle destruction with signs of fibrosis. The autoimmune nature of the disease was shown by high titers of antimyosin antibodies in the sera and IgG deposits in sick heart muscles, as well as focal neutrophil, T cell, and macrophage infiltration of the heart muscle. The sera of the sick mice showed a granular staining pattern on sections of healthy heart muscle. Quantitative analyses of DCM-specific gene expression studies revealed that sets of genes are involved in inflammation, hypoxia, and fibrosis. Treatment with FTY720 (Fingolimod/Gilenya) protected animals from the development of cardiomyopathy. HLA-DQ8(BALB/c)-Tg mice represent a spontaneous autoimmune myocarditis model that may provide a useful tool for studying the autoimmune mechanism of DCM and testing immunosuppressive drugs.

The role of Src protein in the process formation of PC12 cells induced by the proteasome inhibitor MG-132.

The PC12 (rat pheochromocytoma) cell line is a popular model system to study neuronal differentiation. Upon prolonged nerve growth factor (NGF) exposure these tumor cells stop to divide, become polygonal, grow projections and start to look and behave like sympathetic neurons. Differentiation of PC12 cells can also be induced by peptidyl-aldehyde proteasome inhibitors, such as Z-Leu-Leu-Leu-al (also known as MG-132) or via infection of the cells with Rous sarcoma virus. The signal transduction pathways underlying process formation, however, are still not fully understood. The liganded NGF receptor initiates a protein kinase cascade a member of which is Extracellular Signal-Regulated Kinase (ERK). Active ERK1/2 enzymes phosphorylate various cytoplasmic proteins and can also be translocated into the nucleus, where they regulate gene expression by activating key transcription factors. Using immunological methods we detected phosphorylation of TrkA, prolongedactivation of Src, and ERK1/2 with nuclear translocation of the latter during MG-132-induced process formation of PC12 cells. Activated Src remained predominantly cytoplasmic. MG-132-induced sustained ERK1/2 activation, nuclear translocation and neuritogenesis required the intact function of Src since these phenomena were markedly reduced or failed upon chemical inhibition of Src tyrosine protein kinase activity.

Partial rescue of geldanamycin-induced TrkA depletion by a proteasome inhibitor in PC12 cells.

In this work we tried to identify mechanisms that could explain how chemical inhibition of heat-shock protein 90 reduces nerve growth factor signaling in rat pheochromocytoma PC12 cells. Geldanamycin is an antibiotic originally discovered based on its ability to bind heat-shock protein 90. This interaction can lead to the disruption of heat-shock protein 90-containing multimolecular complexes. It can also induce the inhibition or even degradation of partner proteins dissociated from the 90 kDa chaperone and, eventually, can cause apoptosis, for instance, in PC12 cells. Before the onset of initial apoptotic events, however, a marked decrease in the activity of extracellular signal-regulated kinases ERK 1/2 and protein kinase B/Akt can be observed together with reduced expression of the high affinity nerve growth factor receptor, tropomyosine-related kinase, TrkA, in this cell type. The proteasome inhibitor MG-132 can effectively counteract the geldanamycin-induced reduction of TrkA expression and it can render TrkA and ERK1/2 phosphorylation but not that of protein kinase B/Akt by nerve growth factor again inducible. We have found altered intracellular distribution of TrkA in geldanamycin-treated and proteasome-inhibited PC12 cells that may, at least from the viewpoint of protein localization explain why nerve growth factor remains without effect on protein kinase B/Akt. The lack of protein kinase B/Akt stimulation by nerve growth factor in turn reveals why nerve growth factor treatment cannot save PC12 cells from geldanamycin-induced programmed cell death. Our observations can help to better understand the mechanism of action of geldanamycin, a compound with strong human therapeutical potential.

Impaired activation-induced cell death promotes spontaneous arthritis in antigen (cartilage proteoglycan)-specific T cell receptor-transgenic mice.

OBJECTIVE: To investigate whether genetic preponderance of a T cell receptor (TCR) recognizing an arthritogenic peptide of human cartilage proteoglycan (PG) is sufficient for development of arthritis. METHODS: We performed a longitudinal study using BALB/c mice expressing a TCR that recognizes the arthritogenic ATEGRVRVNSAYQDK peptide of human cartilage PG. PG-specific TCR-transgenic (PG-TCR-Tg) mice were inspected weekly for peripheral arthritis until 12 months of age. Peripheral joints were examined histologically, and T cell responses, T cell activation markers, serum cytokines, and autoantibodies were measured. Apoptosis and signaling studies were performed in vitro on T cells from aged PG-TCR-Tg mice. RESULTS: Spontaneous arthritis developed as early as 5-6 months of age, and the incidence increased to 40-50% by 12 months of age. Progressive inflammation began with cartilage and bone erosions in the interphalangeal joints, and later expanded to the proximal joints of the front and hind paws. Spontaneous arthritis was associated with a high proportion of activated CD4+ T cells, enhanced interferon-γ and interleukin-17 (IL-17) production, and elevated levels of serum autoantibodies. PG-TCR-Tg mice lacking IL-4 developed arthritis earlier and at a higher incidence than IL-4-sufficient mice. Antigen-specific activation-induced cell death was diminished in vitro in CD4+ T cells of PG-TCR-Tg mice with spontaneous arthritis, especially in those lacking IL-4. CONCLUSION: The presence of CD4+ T cells expressing a TCR specific for an arthritogenic PG epitope is sufficient to trigger spontaneous autoimmune inflammation in the joints of BALB/c mice. IL-4 appears to be a negative regulator of this disease, through attenuation of activation-induced cell death.

Age-related changes in arthritis susceptibility and severity in a murine model of rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) most often begins in females in the fourth-fifth decade of their life, suggesting that the aging of the immune system (immunosenescence) has a major role in this disease. Therefore, in the present study, we sought to investigate the effect of age on arthritis susceptibility in BALB/c mice using the proteoglycan (PG)-induced arthritis (PGIA) model of RA. RESULTS: We have found that young, 1-month-old female BALB/c mice are resistant to the induction of PGIA, but with aging they become susceptible. PG-induced T cell responses decline with age, whereas there is a shift toward Th1 cytokines. An age-dependent decrease in T cell number is associated with an increased ratio of the memory phenotype, and lower CD28 expression. Antigen-presenting cells shifted from macrophages and myeloid dendritic cells in young mice toward B cells in older mice. The regulatory/activated T cell ratio decreases in older mice after PG injections indicating impaired regulation of the immune response. CONCLUSION: We conclude that immunosenescence could alter arthritis susceptibility in a very complex manner including both adaptive and innate immunities, and it cannot be determined by a single trait. Cumulative alterations in immunoregulatory functions closely resemble human disease, which makes this systemic autoimmune arthritis model of RA even more valuable.

Th1/Th17 polarization and acquisition of an arthritogenic phenotype in arthritis-susceptible BALB/c, but not in MHC-matched, arthritis-resistant DBA/2 mice.

Proteoglycan (PG) aggrecan-induced arthritis (PGIA) is a murine model of rheumatoid arthritis (RA). Although BALB/c and DBA/2 mice share the same MHC (H-2d) haplotype, the BALB/c strain is susceptible to PGIA, while DBA/2 mice are resistant. Therefore, these two inbred mouse strains provide an opportunity to study arthritis susceptibility factors excluding the effects of MHC-associated genetic components. The goal of this study was to monitor changes in the cellular composition and activation state following intra-peritoneal (i.p.) immunization to induce PGIA; additionally, we sought to identify new susceptibility factors by comparing PG-induced immune responses in BALB/c and DBA/2 mice. Upon i.p. PG injection, resident naive B1 cells are replaced by both T cells and conventional B cells in the peritoneum of BALB/c mice. These peritoneal T cells produce IFNgamma and IL-17, cytokines shown to be important in RA and corresponding arthritis models. Moreover, peritoneal cells can adoptively transfer PGIA to SCID mice, demonstrating their arthritogenic properties. Our results indicate that repeatedly injected antigen leads to the recruitment and activation of immune cells in the peritoneum; these cells then trigger the effector phase of the disease. The migration and activation of T(h)1/T(h)17 cells in the peritoneal cavity in response to PG immunization, which did not occur in the arthritis-resistant DBA/2 strain, may be critical factors of arthritis susceptibility in BALB/c mice.

BALB/c mice genetically susceptible to proteoglycan-induced arthritis and spondylitis show colony-dependent differences in disease penetrance.

INTRODUCTION: The major histocompatibility complex (H-2d) and non-major histocompatibility complex genetic backgrounds make the BALB/c strain highly susceptible to inflammatory arthritis and spondylitis. Although different BALB/c colonies develop proteoglycan-induced arthritis and proteoglycan-induced spondylitis in response to immunization with human cartilage proteoglycan, they show significant differences in disease penetrance despite being maintained by the same vendor at either the same or a different location. METHODS: BALB/c female mice (24 to 26 weeks old after 4 weeks of acclimatization) were immunized with a suboptimal dose of cartilage proteoglycan to explore even minute differences among 11 subcolonies purchased from five different vendors. In vitro-measured T-cell responses, and serum cytokines and (auto)antibodies were correlated with arthritis (and spondylitis) phenotypic scores. cDNA microarrays were also performed using spleen cells of naïve and immunized BALB/cJ and BALB/cByJ mice (both colonies from The Jackson Laboratory, Bar Harbor, ME, USA), which represent the two major BALB/c sublines. RESULTS: The 11 BALB/c colonies could be separated into high (n = 3), average (n = 6), and low (n = 2) responder groups based upon their arthritis scores. While the clinical phenotypes showed significant differences, only a few immune parameters correlated with clinical or histopathological abnormalities, and seemingly none of them affected differences found in altered clinical phenotypes (onset time, severity or incidence of arthritis, or severity and progression of spondylitis). Affymetrix assay (Affymetrix, Santa Clara, CA, USA) explored 77 differentially expressed genes (at a significant level, P < 0.05) between The Jackson Laboratory's BALB/cJ (original) and BALB/cByJ (transferred from the National Institutes of Health, Bethesda, MD, USA). Fourteen of the 77 differentially expressed genes had unknown function; 24 of 77 genes showed over twofold differences, and only 8 genes were induced by immunization, some in both colonies. CONCLUSIONS: Using different subcolonies of the BALB/c strain, we can detect significant differences in arthritis phenotypes, single-nucleotide polymorphisms (SNPs), and a large number of differentially expressed genes, even in non-immunized animals. A number of the known genes (and SNPs) are associated with immune responses and/or arthritis in this genetically arthritis-prone murine strain, and a number of genes of as-yet-unknown function may affect or modify clinical phenotypes of arthritis and/or spondylitis.