Altered Subcellular Localization of Heat Shock Protein 90 Is Associated with Impaired Expression of the Aryl Hydrocarbon Receptor Pathway in Dogs

The aryl hydrocarbon receptor (AHR) mediates biological responses to toxic chemicals. An unexpected role for AHR in vascularization was suggested when mice lacking AHR displayed impaired closure of the ductus venosus after birth, as did knockout mice for aryl hydrocarbon receptor interacting protein (AIP) and aryl hydrocarbon receptor nuclear translocator (ARNT). The resulting intrahepatic portosystemic shunts (IHPSS) are frequently diagnosed in specific dog breeds, such as the Irish wolfhound. We compared the expression of components of the AHR pathway in healthy Irish wolfhounds and dogs with IHPSS. To this end, we analyzed the mRNA expression in the liver of AHR,AIP, ARNT, and other genes involved in this pathway, namely, those for aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), hypoxia inducible factor 1alpha (HIF1A), heat shock protein 90AA1 (HSP90AA1), cytochromes P450 (CYP1A1, CYP1A2, and CYP1B1), vascular endothelial growth factor A (VEGFA), nitric oxide synthesase 3 (NOS3), and endothelin (EDN1). The observed low expression of AHR mRNA in the Irish wolfhounds is in associated with a LINE-1 insertion in intron 2, for which these dogs were homozygous. Down regulation in Irish wolfhounds was observed for AIP, ARNT2, CYP1A2, CYP1B1 and HSP90AA1 expression, whereas the expression of HIF1A was increased. Immunohistochemistry revealed lower levels of AHR, HIF1A, and VEGFA protein in the nucleus and lower levels of ARNT and HSP90AA1 protein in the cytoplasm of the liver cells of Irish wolfhounds. The impaired expression of HSP90AA1 could trigger the observed differences in mRNA and protein levels and therefore explain the link between two very different functions of AHR: regulation of the closure of the ductus venosus and the response to toxins.     

A novel DCAF1‐binding motif required for Vpx‐mediated degradation of nuclear SAMHD1 and Vpr‐induced G2 arrest

HIV‐2 and closely related SIV Vpx proteins are essential for viral replication in macrophages and dendritic cells. Vpx hijacks DCAF1–DDB1–Cul4 E3 ubiquitin ligase to promote viral replication. DCAF1 is essential for cell proliferation and embryonic development and is responsible for the polyubiquitination of poorly defined cellular proteins. How substrate receptors recruit the DCAF1‐containing E3 ubiquitin ligase to induce protein degradation is still poorly understood. Here we identify a highly conserved motif (Wx4Φx2Φx3AΦxH) that is present in diverse Vpx and Vpr proteins of primate lentiviruses. We demonstrate that the Wx4Φx2Φx3AΦxH motif in SIVmac Vpx is required for both the Vpx–DCAF1 interaction and/or Vpx‐mediated degradation of SAMHD1. DCAF1‐binding defective Vpx mutants also have impaired ability to promote SIVΔVpx virus infection of myeloid cells. Critical amino acids in the Wx4Φx2Φx3AΦxH motif of SIV Vpx that are important for DCAF1 interaction maintained the ability to bind SAMHD1, indicating that the DCAF1 and SAMHD1 interactions involve distinctive interfaces in Vpx. Surprisingly, VpxW24A mutant proteins that were still capable of binding DCAF1 and SAMHD1 lost the ability to induce SAMHD1 degradation, suggesting that Vpx is not a simple linker between the DCAF1–DDB1–Cul4 E3 ubiquitin ligase and its substrate, SAMHD1.VpxW24A maintained the ability to accumulate in the nucleus despite the fact that nuclear, but not cytoplasmic, mutant forms of SAMHD1 were more sensitive to Vpx‐mediated degradation. The Wx4Φx2Φx3AΦxH motif in HIV‐1 Vpr is also required for the Vpr–DCAF1 interaction and Vpr‐induced G2 cell cycle arrest. Thus, our data reveal previously unrecognized functional interactions involved in the assembly of virally hijacked DCAF1–DDB1‐based E3 ubiquitin ligase complex.     

乏氧诱导因子结构、表达及调控

乏氧诱导因子（HIF）是乏氧应答中起重要作用的转录因子,一直是乏氧研究的焦点.HIF由α亚基和β亚基组成,α亚基包括HIF-1α、HIF-2α和HIF-3α,其中α亚基因诱导条件不同通过选择性剪接产生不同变体.β亚基包括ARNT、ARNT2和ARNT3.α与β亚基在乏氧等应激反应时形成二聚体HIF启动靶基因转录表达,参与多种细胞生物学功能的调控.目前为止,大多数的研究都集中于野生型HIF-1α,对它的结构、表达调控及其调控做了相对全面而清楚的了解.后来通过多种策略及方法,陆续发现并克隆出了除HIF-1α外的HIF各亚基.研究不再局限于HIF-1α,而是扩展至HIF整个系统,如相继发现的HIF-2α和HIF-3α亚基,以及它们的变体,对HIF-1α的研究也更深入了,但是关于HIF-1α的变体、HIF-2α、HIF－3α及β亚基的表达调控及功能还不明确,是未来研究的方向.本文全面介绍HIF的最新研究进展,阐述HIF各亚基的结构、表达调控及其靶基因的表达情况.     

Aryl hydrocarbon receptor-mediated inhibition of LNCaP prostate cancer cell growth and hormone-induced transactivation

LNCaP prostate cancer cells express the aryl hydrocarbon receptor (AhR), and treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and an Ah-responsive reporter gene. Similar results were obtained with the selective AhR modulator 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF); however, TCDD but not 6-MCDF induced degradation of the AhR protein. TCDD and 6-MCDF inhibited growth of LNCaP cells, and inhibitory AhR-androgen receptor (AR) crosstalk was investigated in cells transfected with constructs containing the androgen-responsive probasin promoter (-288 to +28) (pPB) or three copies of the -244 to -96 region of this promoter (pARR(3)). Ten nanomolar dihydrotestosterone (DHT) and 17 beta-estradiol (E2) induced transactivation in LNCaP cells transfected with pPB or pARR(3); however, inhibitory AhR-AR crosstalk was observed only with the latter construct. 6-MCDF and TCDD did not inhibit DHT- or E2-induced transactivation in ZR-75 human breast cancer cells, indicating that these interactions were promoter and cell context-dependent. Both E2 and DHT stabilized AR protein in LNCaP cells; however, cotreatment with TCDD or 6-MCDF decreased AR protein levels. These results indicate that inhibitory AhR-AR crosstalk in prostate cancer cells is complex and for some responses, AR protein stability may play a role.     

Crosstalk between estrogen receptor alpha and the aryl hydrocarbon receptor in breast cancer cells involves unidirectional activation of proteasomes

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxin that activates the aryl hydrocarbon receptor (AhR) and disrupts multiple endocrine signaling pathways. T47D human breast cancer cells express a functional estrogen receptor alpha (ERalpha) and AhR, and treatment of these cells with 17beta-estradiol (E2) or TCDD resulted in a rapid proteasome-dependent decrease in immunoreactive ERalpha and AhR proteins (>60-80%), respectively. E2 did not affect the AhR, whereas TCDD induced proteasome-dependent degradation of both the AhR and ERalpha in T47D and MCF-7 human breast cancer cells, and these responses were specifically blocked by proteasome inhibitors. Thus, TCDD-induced degradation of ERalpha may contribute to the antiestrogenic activity of AhR agonists and this pathway may be involved in AhR-mediated disruption of other endocrine responses.     

Norisoboldine,an Anti-Arthritis Alkaloid Isolated from Radix Linderae,Attenuates Osteoclast Differentiation and Inflammatory Bone Erosion in an Aryl Hydrocarbon Receptor-Dependent Manner

Norisoboldine (NOR), the primary isoquinoline alkaloid constituent of the root of Lindera aggregata, has previously been demonstrated to attenuate osteoclast (OC) differentiation. Accumulative evidence has shown that aryl hydrocarbon receptor (AhR) plays an important role in regulating the differentiation of various cells, and multiple isoquinoline alkaloids can modulate AhR. In the present study, we explored the role of NOR in the AhR signaling pathway. These data showed that the combination of AhR antagonist resveratrol (Res) or α-naphthoflavone (α-NF) nearly reversed the inhibition of OC differentiation through NOR. NOR could stably bind to AhR, up-regulate the nuclear translocation of AhR, and enhance the accumulation of the AhR-ARNT complex, AhR-mediated reporter gene activity and CYP1A1 expression in RAW 264.7 cells, suggesting that NOR might be an agonist of AhR. Moreover, NOR inhibited the nuclear translocation of NF-κB-p65, resulting in the evident accumulation of the AhR-NF-κB-p65 complex, which could be markedly inhibited through either Res or α-NF. Although NOR only slightly affected the expression of HIF-1α, NOR markedly reduced VEGF mRNA expression and ARNT-HIF-1α complex accumulation. In vivo studies indicated that NOR decreased the number of OCs and ameliorated the bone erosion in the joints of rats with collagen-induced arthritis, accompanied by the up-regulation of CYP1A1 and the down-regulation of VEGF mRNA expression in the synovium of rats. A combination of α-NF nearly completely reversed the effects of NOR. In conclusion, NOR attenuated OC differentiation and bone erosion through the activation of AhR and the subsequent inhibition of both NF-κB and HIF pathways.     

HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1

The sterile alpha motif and HD domain-containing protein-1 (SAMHD1) inhibits infection of myeloid cells by human and related primate immunodeficiency viruses (HIV and SIV). This potent inhibition is counteracted by the Vpx accessory virulence factor of HIV-2/SIVsm viruses, which targets SAMHD1 for proteasome-dependent degradation, by reprogramming cellular CRL4(DCAF1) E3 ubiquitin ligase. However, the precise mechanism of Vpx-dependent recruitment of human SAMHD1 onto the ligase, and the molecular interfaces on the respective molecules have not been defined. Here, we show that human SAMHD1 is recruited to the CRL4(DCAF1-Vpx) E3 ubiquitin ligase complex by interacting with the DCAF1 substrate receptor subunit in a Vpx-dependent manner. No stable association is detectable with DCAF1 alone. The SAMHD1 determinant for the interaction is a short peptide located distal to the SAMHD1 catalytic domain and requires the presence of Vpx for stable engagement. This peptide is sufficient to confer Vpx-dependent recruitment to CRL4(DCAF1) and ubiquitination when fused to heterologous proteins. The precise amino acid sequence of the peptide diverges among SAMHD1 proteins from different vertebrate species, explaining selective down-regulation of human SAMHD1 levels by Vpx. Critical amino acid residues of SAMHD1 and Vpx involved in the DCAF1-Vpx-SAMDH1 interaction were identified by mutagenesis. Our findings show that the N terminus of Vpx, bound to DCAF1, recruits SAMHD1 via its C terminus to CRL4, in a species-specific manner for proteasomal degradation.