Coactivation of GR and NFKB alters the repertoire of their binding sites and target genes.

Glucocorticoid receptor (GR) exerts anti-inflammatory action in part by antagonizing proinflammatory transcription factors such as the nuclear factor kappa-b (NFKB). Here, we assess the crosstalk of activated GR and RELA (p65, major NFKB component) by global identification of their binding sites and target genes. We show that coactivation of GR and p65 alters the repertoire of regulated genes and results in their association with novel sites in a mutually dependent manner. These novel sites predominantly cluster with p65 target genes that are antagonized by activated GR and vice versa. Our data show that coactivation of GR and NFKB alters signaling pathways that are regulated by each factor separately and provide insight into the networks underlying the GR and NFKB crosstalk.

Genome-wide interplay of nuclear receptors with the epigenome.

The nuclear receptor superfamily consists of DNA binding transcription factors that are involved in regulating a wide variety of processes such as metabolism, development, reproduction, and immune responses. Upon binding, nuclear receptors modulate transcription through affecting the local chromatin environment via recruitment of various coregulatory proteins. The recent development of new high-throughput sequencing methods allowed for the first time the comprehensive examination of nuclear receptor action in the context of the epigenome. Here, we discuss how recent genome-wide analyses have provided important new insights on the interplay of nuclear receptors and the epigenome in health and disease. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

Gene expression profiling of canine osteosarcoma reveals genes associated with short and long survival times.

BACKGROUND: Gene expression profiling of spontaneous tumors in the dog offers a unique translational opportunity to identify prognostic biomarkers and signaling pathways that are common to both canine and human. Osteosarcoma (OS) accounts for approximately 80% of all malignant bone tumors in the dog. Canine OS are highly comparable with their human counterpart with respect to histology, high metastatic rate and poor long-term survival. This study investigates the prognostic gene profile among thirty-two primary canine OS using canine specific cDNA microarrays representing 20,313 genes to identify genes and cellular signaling pathways associated with survival. This, the first report of its kind in dogs with OS, also demonstrates the advantages of cross-species comparison with human OS. RESULTS: The 32 tumors were classified into two prognostic groups based on survival time (ST). They were defined as short survivors (dogs with poor prognosis: surviving fewer than 6 months) and long survivors (dogs with better prognosis: surviving 6 months or longer). Fifty-one transcripts were found to be differentially expressed, with common upregulation of these genes in the short survivors. The overexpressed genes in short survivors are associated with possible roles in proliferation, drug resistance or metastasis. Several deregulated pathways identified in the present study, including Wnt signaling, Integrin signaling and Chemokine/cytokine signaling are comparable to the pathway analysis conducted on human OS gene profiles, emphasizing the value of the dog as an excellent model for humans. CONCLUSION: A molecular-based method for discrimination of outcome for short and long survivors is useful for future prognostic stratification at initial diagnosis, where genes and pathways associated with cell cycle/proliferation, drug resistance and metastasis could be potential targets for diagnosis and therapy. The similarities between human and canine OS makes the dog a suitable pre-clinical model for future 'novel' therapeutic approaches where the current research has provided new insights on prognostic genes, molecular pathways and mechanisms involved in OS pathogenesis and disease progression.

Transactivation of a growth hormone (GH) promoter-luciferase construct in canine mammary cells.

The gene encoding growth hormone (GH) is expressed not only in the pituitary but also in a variety of non-pituitary tissues. In the female dog, progestins are known to stimulate GH expression in the mammary gland. In order to investigate the regulation of the GH gene expression in the mammary gland, we transfected the canine mammary tumor cell line CMT-U229 with 3 different canine GH promoter-luciferase constructs. The constructs, varying in length between 252 bp and 673 bp, were transfected followed by an incubation for 4 h, 24 h and 48 h with cAMP, all-trans-retinoic acid (RA), 3,3',5-triiodothyronine (T3), 1,25-dihydroxy-vitamin D (VitD), progesterone and EGF. Promoter activity was stimulated by cAMP, T3 and RA whereas VitD clearly inhibited gene expression. However, despite the presence of nuclear and membrane receptors for progesterone, no direct effects of progesterone on promoter activity could be demonstrated. It is concluded that progesterone alone has no direct stimulatory effect on GH transcription. This finding is discussed in relation to the slow onset of progesterone-stimulated GH release in vivo and the absence of Pit-1 in canine mammary tissue.

Role of progestin-induced mammary-derived growth hormone in the pathogenesis of cystic endometrial hyperplasia in the bitch.

Endogenous progesterone and synthetic progestins may induce hypersecretion of growth hormone (GH) of mammary origin, hyperplastic ductular changes in the mammary gland, and the development of cystic endometrial hyperplasia (CEH) in dogs. It was investigated whether progestin-induced mammary GH plays a role in the pathogenesis of CEH in the bitch. During 1 year, bitches with surgically excised mammary glands and healthy control bitches received medroxyprogesterone acetate (MPA). Before and after MPA treatment, uterine and mammary tissues were collected for histological, immunohistochemical, and RT-PCR examination. After MPA administration, the mammary tissue in the control dogs had differentiated into lobulo-alveolar structures and CEH was present in all uteri of both dog groups. In the MPA-exposed mammary tissue of the control dogs, GH could only be demonstrated immunohistochemically in proliferating epithelium. After treatment with MPA the dogs of both groups had immunohistochemically demonstrable GH in the cytoplasm of hyperplastic glandular uterine epithelial cells. RT-PCR analysis of the mammary gland tissue after MPA administration demonstrated a significant higher GH gene, and lower GHR gene expression than before treatment. In the uterus, the expression of the gene encoding for GH was significantly increased in the mastectomized dogs, whereas in the control dogs the expression of the gene encoding for insulin-like growth factor-I had significantly increased with MPA administration. MPA treatment significantly down regulated PR gene expression in the uterus in both dog groups. These results indicate that progestin-induced GH of mammary origin is not an essential component in the development of CEH in the bitch.

Ligand-independent canonical Wnt activity in canine mammary tumor cell lines associated with aberrant LEF1 expression.

Pet dogs very frequently develop spontaneous mammary tumors and have been suggested as a good model organism for breast cancer research. In order to obtain an insight into underlying signaling mechanisms during canine mammary tumorigenesis, in this study we assessed the incidence and the mechanism of canonical Wnt activation in a panel of 12 canine mammary tumor cell lines. We show that a subset of canine mammary cell lines exhibit a moderate canonical Wnt activity that is dependent on Wnt ligands, similar to what has been described in human breast cancer cell lines. In addition, three of the tested canine mammary cell lines have a high canonical Wnt activity that is not responsive to inhibitors of Wnt ligand secretion. Tumor cell lines with highly active canonical Wnt signaling often carry mutations in key members of the Wnt signaling cascade. These cell lines, however, carry no mutations in the coding regions of intracellular Wnt pathway components (APC, ß-catenin, GSK3ß, CK1α and Axin1) and have a functional ß-catenin destruction complex. Interestingly, however, the cell lines with high canonical Wnt activity specifically overexpress LEF1 mRNA and the knock-down of LEF1 significantly inhibits TCF-reporter activity. In addition, LEF1 is overexpressed in a subset of canine mammary carcinomas, implicating LEF1 in ligand-independent activation of canonical Wnt signaling in canine mammary tumors. We conclude that canonical Wnt activation may be a frequent event in canine mammary tumors both through Wnt ligand-dependent and novel ligand-independent mechanisms.