Subcellular localization of the aryl hydrocarbon receptor is modulated by the immunophilin homolog hepatitis B virus X-associated protein 2

The hepatitis B virus X-associated protein 2 (XAP2) is an immunophilin homolog and core component of the aryl hydrocarbon receptor (AhR). Immunophilins are components of many steroid receptor complexes, serving a largely unknown function. Transiently expressed AhR.YFP (yellow fluorescent protein) localized to the nuclei of COS-1 and NIH-3T3 cells. Co-expression of AhR.YFP with XAP2 restored cytoplasmic localization, which was reversed by 2,3,7, 8-tetrachlorodibenzo-p-dioxin treatment (TCDD). The effect of XAP2 on AhR localization was specific involving a nuclear localization signal-mediated pathway. Examination of the ratio of AhR to XAP2 in the AhR complex revealed that approximately 25% of transiently expressed AhR was associated with XAP2, in contrast with approximately 100% when the AhR and XAP2 were co-expressed. Strikingly, TCDD did not influence these ratios, suggesting that ligand binding initiates nuclear translocation prior to complex dissociation. Analysis of endogenous AhR in Hepa-1 cells revealed that approximately 40% of the AhR complex was associated with XAP2, predicting observed AhR localization to cytoplasm and nuclei. This study reveals a novel functional role for the immunophilin-like component of a soluble receptor complex and provides new insight into the mechanism of AhR-mediated signal transduction, demonstrating the existence of two structurally distinct and possibly functionally unique forms of the AhR.     

Impact of aryl hydrocarbon receptor (AhR) knockdown on cell cycle progression in human HaCaT keratinocytes

Abstract While activation of the aryl hydrocarbon receptor (AhR) by exogenous ligands is well investigated, its physiological function is less understood. By extending research in AhR biology, evidence appeared that the receptor generally plays an important role in cell physiology. In keratinocytes, little is known about endogenous functions of the AhR. In order to expand this knowledge, we analyzed the impact of AhR knockdown on cell cycle progression in HaCaT cells and showed that proliferation of siAhR HaCaT cells was significantly decreased. In line with that result, western blot analysis revealed that protein level of the cyclin dependent kinase inhibitor p27(KIP1) was increased, whereas protein level of the cyclin dependent kinase (CDK) 2 was reduced. CDK4 and CDK6 protein levels remained unchanged, whereas protein level of the retinoblastoma protein (pRB) was reduced. By measuring ethoxyresorufin-O-deethylase (EROD) activity we showed that endogenous cytochrome P450 1 (CYP1), especially CYP1A1 is required for normal cell cycle in HaCaT cells, as well. To the best of our knowledge, we provide evidence for the first time in human skin cells, that in the absence of exogenous ligands, the AhR promotes cell cycle progression in HaCaT cells and one can speculate that this is the physiological function of this receptor in keratinocytes.     

The impact of aryl hydrocarbon receptor signaling on matrix metabolism: implications for development and disease

The aryl hydrocarbon receptor (AhR) was identified as the receptor for polycyclic aromatic hydrocarbons and related compounds. However, novel data indicate that the AhR binds a variety of unrelated endogenous and exogenous compounds. Although AhR knockout mice demonstrate that this receptor has a role in normal development and physiology, the function of this receptor is still unclear. Recent evidence suggests that AhR signaling also alters the expression of genes involved in matrix metabolism, specifically the matrix metalloproteinases (MMPs). MMP expression and activity is critical to normal physiological processes that require tissue remodeling, as well as in mediating the progression of a variety of diseases. MMPs not only degrade structural proteins, but are also important mediators of cell signaling near or at the cell membrane through exposure of cryptic sites, release of growth factors, and cleavage of receptors. Therefore, AhR modulation of MMP expression and activity may be critical, not only in pathogenesis, but also in understanding the endogenous function of the AhR. In this review we will examine the data indicating a role for the AhR-signaling pathway in the regulation of matrix remodeling, and discuss potential molecular mechanisms.     

Aryl hydrocarbon receptor catabolic activity in bone metabolism is osteoclast dependent in vivo

Bone mass is regulated by various molecules including endogenous factors as well as exogenous factors, such as nutrients and pollutants. Aryl hydrocarbon receptor (AhR) is known as a dioxin receptor and is responsible for various pathological and physiological processes. However, the role of AhR in bone homeostasis remains elusive because the cell type specific direct function of AhR has never been explored in vivo. Here, we show the cell type specific function of AhR in vivo in bone homeostasis. Systemic AhR knockout (AhRKO) mice exhibit increased bone mass with decreased resorption and decreased formation. Meanwhile, osteoclast specific AhRKO (AhR^ΔOc/ΔOc) mice have increased bone mass with reduced bone resorption, although the mice lacking AhR in osteoblasts have a normal bone phenotype. Even under pathological conditions, AhR^ΔOc/ΔOc mice are resistant to sex hormone deficiency-induced bone loss resulting from increased bone resorption. Furthermore, 3-methylcholanthrene, an AhR agonist, induces low bone mass with increased bone resorption in control mice, but not in AhR^ΔOc/ΔOc mice. Taken together, cell type specific in vivo evidence for AhR functions indicates that osteoclastic AhR plays a significant role in maintenance of bone homeostasis, suggesting that inhibition of AhR in osteoclasts can be beneficial in the treatment of osteoporosis.     

The aromatic hydrocarbon receptor modulates the Hepa 1c1c7 cell cycle and differentiated state independently of dioxin.

The aromatic hydrocarbon receptor (AhR) has been defined and characterized according to its ability to mediate biological responses to exogenous ligands, such as the synthetic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The natural ligand(s) for AhR is unknown, and we know relatively little about AhR function in the absence of TCDD. Here, we have exploited the availability of AhR-defective (AhR-D) mouse hepatoma (Hepa 1c1c7) cells to analyze AhR's effects under conditions in which TCDD is not present. Our results reveal that AhR-D cells exhibit a different morphology, decreased albumin synthesis, and a prolonged doubling time compared with wild-type cells. Introduction of AhR cDNA into AhR-D cells by stable transfection alters these characteristics such that the cells resemble wild-type cells. Conversely, introduction of antisense AhR cDNA into wild-type cells changes their phenotype such that they resemble AhR-D cells. Fluorescence microscopy reveals that AhR-D cells do not exhibit an increased rate of death. Flow cytometric and biochemical analyses imply that the slowed growth rate of AhR-D cells reflects prolongation of G1. Our findings reveal a potential link between AhR and the G1 phase of the Hepa 1c1c7 cell cycle. These effects of AhR occur in the absence of TCDD. We speculate that they represent responses to an endogenous AhR ligand in Hepa 1c1c7 cells.