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Abstract Background Early studies suggested that TR4 nuclear receptor might play important roles in the skeletal development, yet its detailed mechanism remains unclear. Methods We generated TR4 knockout mice and compared skeletal development with their wild type littermates. Primary bone marrow cells were cultured and we assayed bone differentiation by alkaline phosphatase and alizarin red staining. Primary calvaria were cultured and osteoblastic marker genes were detected by quantitative PCR. Luciferase reporter assays, chromatin immunoprecipitation (ChIP) assays, and electrophoretic mobility shift assays (EMSA) were performed to demonstrate TR4 can directly regulate bone differentiation marker osteocalcin. Results We first found mice lacking TR4 might develop osteoporosis. We then found that osteoblast progenitor cells isolated from bone marrow of TR4 knockout mice displayed reduced osteoblast differentiation capacity and calcification. Osteoblast primary cultures from TR4 knockout mice calvaria also showed higher proliferation rates indicating lower osteoblast differentiation ability in mice after loss of TR4. Mechanism dissection found the expression of osteoblast markers genes, such as ALP, type I collagen alpha 1, osteocalcin, PTH, and PTHR was dramatically reduced in osteoblasts from TR4 knockout mice as compared to those from TR4 wild type mice. In vitro cell line studies with luciferase reporter assay, ChIP assay, and EMSA further demonstrated TR4 could bind directly to the promoter region of osteocalcin gene and induce its gene expression at the transcriptional level in a dose dependent manner. Conclusions Together, these results demonstrate TR4 may function as a novel transcriptional factor to play pathophysiological roles in maintaining normal osteoblast activity during the bone development and remodeling, and disruption of TR4 function may result in multiple skeletal abnormalities.

Acute hepatopancreatic necrosis disease (AHPND) in shrimp is caused by Vibrio strains that harbor a pVA1-like plasmid containing the pirA and pirB genes. It is also known that the production of the PirA and PirB proteins, which are the key factors that drive the observed symptoms of AHPND, can be influenced by environmental conditions and that this leads to changes in the virulence of the bacteria. However, to our knowledge, the mechanisms involved in regulating the expression of the pirA/pirB genes have not previously been investigated. In this study, we show that in the AHPND-causing Vibrio parahaemolyticus 3HP strain, the pirAvp and pirBvp genes are highly expressed in the early log phase of the growth curve. Subsequently, the expression of the PirAvp and PirBvp proteins continues throughout the log phase. When we compared mutant strains with a deletion or substitution in two of the quorum sensing (QS) master regulators, luxO and/or opaR (luxOD47E, ΔopaR, ΔluxO, and ΔopaRΔluxO), our results suggested that expression of the pirAvp and pirBvp genes was related to the QS system, with luxO acting as a negative regulator of pirAvp and pirBvp without any mediation by opaRvp. In the promoter region of the pirAvp/pirBvp operon, we also identified a putative consensus binding site for the QS transcriptional regulator AphB. Real-time PCR further showed that aphBvp was negatively controlled by LuxOvp, and that its expression paralleled the expression patterns of pirAvp and pirBvp. An electrophoretic mobility shift assay (EMSA) showed that AphBvp could bind to this predicted region, even though another QS transcriptional regulator, AphAvp, could not. Taken together, these findings suggest that the QS system may regulate pirAvp/pirBvp expression through AphBvp. [ABSTRACT FROM AUTHOR]

Acute hepatopancreatic necrosis disease (AHPND) is a newly emergent penaeid shrimp disease which can cause 70-100% mortality in Penaeus vannamei and Penaeus monodon , and has resulted in enormous economic losses since its appearance. AHPND is caused by the specific strains of Vibrio parahaemolyticus that harbor the pVA1 plasmid and express PirA vp and PirB vp toxins. These two toxins have been reported to form a binary complex. When both are present, they lead to the death of shrimp epithelial cells in the hepatopancreas and cause the typical histological symptoms of AHPND. However, the binding mode of PirA vp and PirB vp has not yet been determined. Here, we used isothermal titration calorimetry (ITC) to measure the binding affinity of PirA vp and PirB vp . Since the dissociation constant ( K d = 7.33 ± 1.20 μM) was considered too low to form a sufficiently stable complex for X-ray crystallographic analysis, we used alternative methods to investigate PirA vp -PirB vp interaction, first by using gel filtration to evaluate the molecular weight of the PirA vp /PirB vp complex, and then by using cross-linking and hydrogen-deuterium exchange (HDX) mass spectrometry to further understand the interaction interface between PirA vp and PirB vp . Based on these results, we propose a heterotetrameric interaction model of this binary toxin complex. This model provides insight of how conformational changes might activate the PirB vp N-terminal pore-forming domain and should be helpful for devising effective anti-AHPND strategies in the future.

In aquaculture, shrimp farming is a popular field. The benefits of shrimp farming include a relatively short grow-out time, high sale price, and good cost recovery. However, outbreaks of serious diseases inflict serious losses, and acute hepatopancreatic necrosis disease (AHPND) is an emerging challenge to this industry. In South American white shrimp ( Penaeus vannamei ) and grass shrimp ( Penaeus monodon ), this disease has a 70-100% mortality. The pathogenic agent of AHPND is a specific strain of Vibrio parahaemolyticus which contains PirA vp and PirB vp toxins encoded in the pVA1 plasmid. PirA vp and PirB vp have been shown to cause the typical histological symptoms of AHPND in infected shrimps, and in this review, we will focus on our structural understanding of these toxins. By analyzing their structures, a possible cytotoxic mechanism, as well as strategies for anti-AHPND drug design, is proposed., Competing Interests: The authors declare no conflict of interest.

Testicular nuclear receptors 2 and 4 (TR2, TR4), also known as NR2C1 and NR2C2, belong to the nuclear receptor superfamily and were first cloned in 1989 and 1994, respectively. Although classified as orphan receptors, several natural molecules, their metabolites, and synthetic compounds including polyunsaturated fatty acids (PUFAs), PUFA metabolites 13-hydroxyoctadecadienoic acid, 15-hydroxyeicosatetraenoic acid, and the antidiabetic drug thiazolidinediones can transactivate TR4. Importantly, many of these ligands/activators can also transactivate peroxisome proliferator-activated receptor gamma (PPARγ), also known as NR1C3 nuclear receptor. Both TR4 and PPARγ can bind to similar hormone response elements (HREs) located in the promoter of their common downstream target genes. However, these two nuclear receptors, even with shared ligands/activators and shared binding ability for similar HREs, have some distinct functions in many diseases they influence. In cancer, PPARγ inhibits thyroid, lung, colon, and prostate cancers but enhances bladder cancer. In contrast, TR4 inhibits liver and prostate cancer initiation but enhances pituitary corticotroph, liver, and prostate cancer progression. In type 2 diabetes, PPARγ increases insulin sensitivity but TR4 decreases insulin sensitivity. In cardiovascular disease, PPARγ inhibits atherosclerosis but TR4 enhances atherosclerosis through increasing foam cell formation. In bone physiology, PPARγ inhibits bone formation but TR4 increases bone formation. Together, the contrasting impact of TR4 and PPARγ on different diseases may raise a critical issue about drug used to target any one of these nuclear receptors., (© 2017 Elsevier Inc. All rights reserved.)