BACKGROUND & AIMS The pathogenesis of liver fibrosis involves activation of hepatic stellate cells, which is associated with depletion of intracellular lipid droplets. When hepatocytes undergo autophagy, intracellular lipids are degraded in lysosomes. We investigated whether autophagy also promotes loss of lipids in hepatic stellate cells to provide energy for their activation and extended these findings to other fibrogenic cells. METHODS We analyzed hepatic stellate cells from C57BL/6 wild-type, Atg7F/F, and Atg7F/F-GFAP-Cre mice, as well as the mouse stellate cell line JS1. Fibrosis was induced in mice using CCl4 or thioacetamide (TAA); liver tissues and stellate cells were analyzed. Autophagy was blocked in fibrogenic cells from liver and other tissues using small interfering RNAs against Atg5 or Atg7 and chemical antagonists. Human pulmonary fibroblasts were isolated from samples of lung tissue from patients with idiopathic pulmonary fibrosis or from healthy donors. RESULTS In mice, induction of liver injury with CCl4 or TAA increased levels of autophagy. We also observed features of autophagy in activated stellate cells within injured human liver tissue. Loss of autophagic function in cultured mouse stellate cells and in mice following injury reduced fibrogenesis and matrix accumulation; this effect was partially overcome by providing oleic acid as an energy substrate. Autophagy also regulated expression of fibrogenic genes in embryonic, lung, and renal fibroblasts. CONCLUSIONS Autophagy of activated stellate cells is required for hepatic fibrogenesis in mice. Selective reduction of autophagic activity in fibrogenic cells in liver and other tissues might be used to treat patients with fibrotic diseases.

The μ and δ types of opioid receptors form heteromers that exhibit pharmacological and functional properties distinct from those of homomeric receptors. To characterize these complexes in the brain, we generated antibodies that selectively recognize the μ-δ heteromer and blocked its in vitro signaling. With these antibodies, we showed that chronic, but not acute, morphine treatment caused an increase in the abundance of μ-δ heteromers in key areas of the central nervous system that are implicated in pain processing. Because of its distinct signaling properties, the μ-δ heteromer could be a therapeutic target in the treatment of chronic pain and addiction.

Opiates are analgesics of choice in the treatment of chronic pain, but their long-term use leads to the development of physiological tolerance. Thus, understanding the mechanisms modulating the response of their receptor, the μ opioid receptor (μOR), is of great clinical relevance. Here we show that heterodimerization of μOR with δ opioid receptors (δOR) leads to a constitutive recruitment of β-arrestin2 to the receptor complex resulting in changes in the spatio-temporal regulation of ERK1/2 signaling. The involvement of β-arrestin2 is further supported by studies using β-arrestin2 siRNA in cells endogenously expressing the heterodimers. The association of β-arrestin2 with the heterodimer can be altered by treatment with a combination of μOR agonist (DAMGO) and δOR antagonist (Tipp Ψ ), and this leads to a shift in the pattern of ERK1/2 phosphorylation to the pattern observed with μOR alone. These data indicate that, in the naive state, μOR-δOR heterodimers are in a conformation conducive to β-arrestin-mediated signaling. Destabilization of this conformation by cotreatment with μOR and δOR ligands leads to a switch to a non-β-arrestin-mediated signaling. Taken together, these results show for the first time that μOR-δOR heterodimers, by differentially recruiting β-arrestin, modulate the spatio-temporal dynamics of opioid receptor signaling.

Background & Aims Metabolic stress during liver injury enhances autophagy and provokes stellate cell activation, with secretion of scar matrix. Conditions that augment protein synthesis increase demands on the endoplasmic reticulum (ER) folding capacity and trigger the unfolded protein response (UPR) to cope with resulting ER stress. Generation of reactive oxygen species (ROS) is a common feature of hepatic fibrogenesis, and crosstalk between oxidative stress and ER stress has been proposed. The aim of our study was to determine the impact of oxidant and ER stress on stellate cell activation. Methods Oxidant stress was induced in hepatic stellate cells using H2O2 in culture or by ethanol feeding in vivo, and the UPR response was analyzed. Because the branch of the UPR mainly affected was IREα, we blocked this pathway in stellate cells and analyzed the fibrogenic response, together with autophagy and downstream MAPK signaling. The Nrf2 antioxidant response was also evaluated in stellate cells under oxidant stress conditions. Results H2O2 treatment in culture or ethanol feeding in vivo increased the UPR response based on splicing of XBP1 mRNA, which triggered autophagy. The Nrf2-mediated antioxidant response, as measured by qRT-PCR of its target genes was also induced under ER stress conditions. Conversely, blockade of the IRE1 pathway in stellate cells significantly decreased both their activation and autophagic activity in a p38 MAPK dependent manner, leading to a reduced fibrogenic response. Conclusions These data implicate mechanisms underlying protein folding quality control in regulating the fibrogenic response in hepatic stellate cells.

Cannabinoid receptor 1 (CB(1)) is an abundant G protein-coupled receptor, involved in a number of physiological processes. This receptor is localized at the plasma membrane, as well as in intracellular vesicles. The trafficking events leading to this intracellular localization remain controversial. In this study, we examine the differential trafficking of CB(1) receptors and its implication on signaling. We find that the transfected tagged receptors are predominantly at the plasma membrane, whereas endogenous receptors exhibit an intracellular localization. We also find that intracellular endogenous CB(1) receptors do not have an endocytic origin. Instead, these receptors associate with the adaptor protein AP-3 and traffic to the lysosomes. siRNA-mediated AP-3delta knockdown leads to enhanced cell surface localization of CB(1) receptors. Finally, we show that CB(1) receptors in the late endosomal/lysosomal compartment are associated with heterotrimeric G proteins and mediate signal transduction. These results suggest that intracellular CB(1) receptors are functional and that their spatial segregation is likely to significantly affect receptor function.

The mechanism of G protein-coupled receptor (GPCR) signal integration is controversial. While GPCR assembly into hetero-oligomers facilitates signal integration of different receptor types, cross-talk between Gai-and Gaqcoupled receptors is often thought to be oligomerization independent. In this study, we examined the mechanism of signal integration between the Gai-coupled type I cannabinoid receptor (CB 1 R) and the Gaq-coupled AT1R. We find that these two receptors functionally interact, resulting in the potentiation of AT1R signalling and coupling of AT1R to multiple G proteins. Importantly, using several methods, that is, co-immunoprecipitation and resonance energy transfer assays, as well as receptor-and heteromerselective antibodies, we show that AT1R and CB 1 R form receptor heteromers. We examined the physiological relevance of this interaction in hepatic stellate cells from ethanol-administered rats in which CB 1 R is upregulated. We found a significant upregulation of AT1R-CB 1 R heteromers and enhancement of angiotensin II-mediated signalling, as compared with cells from control animals. Moreover, blocking CB 1 R activity prevented angiotensin II-mediated mitogenic signalling and profibrogenic gene expression. These results provide a molecular basis for the pivotal role of heteromer-dependent signal integration in pathology.

opioid receptors are G protein-coupled receptors that mediate the pain-relieving effects of clinically used analgesics, such as morphine. Accumulating evidence shows that -␦ opioid heterodimers have a pharmacologic profile distinct from those of the or ␦ homodimers. Because the heterodimers exhibit distinct signaling properties, the protein and mechanism regulating their levels have significant effects on morphine-mediated physiology. We report the characterization of RTP4, a Golgi chaperone, as a regulator of the levels of heterodimers at the cell surface. We show that the association with RTP4 protects -␦ receptors from ubiquitination and degradation. This leads to increases in surface heterodimer levels, thereby affecting signaling. Thus, the oligomeric organization of opioid receptors is controlled by RTP4, and this governs their membrane targeting and functional activity. This work is the first report of the identification of a chaperone involved in the regulation of the biogenesis of a family A GPCR heterodimer. The identification of such factors as RTP4 controlling dimerization will provide insight into the regulation of heterodimers in vivo. This has implications in the modulation of pharmacology of their endogenous ligands, and in the development of drugs with specific therapeutic effects.he dimerization of G protein-coupled receptors (GPCRs) is a widely studied phenomenon that can profoundly modify the pharmacology of interacting partners in vitro. Allosteric modulation of ligand binding, alteration in G protein activation, and coupling to a new signaling pathway are known to result from GPCR association (1, 2). Thus, a greater level of complexity could arise from in vivo receptor-receptor interactions, making dimers promising targets for the development of new drugs with more specific therapeutic effects (3). Early studies using heterologous expression explored the functional outcome resulting from the association of two identical (homodimers) or, in most cases, two distinct receptors (heterodimers). Recent studies have focused on a role for receptor association in their folding and maturation, that is, GPCR oligomer biosynthesis (4). Similar to the dimerization-dependent expression known for class C receptors (5), class A receptors have also been found to require dimerization for enhanced expression (6, 7). In addition, inefficient targeting of GPCR oligomers in vivo has been shown to be the cause of pathophysiology in some cases, thus emphasizing the importance of the proper receptor assemblage for normal delivery to the cell surface (8, 9). The mechanism underlying this phenomenon has not been well explored.The three subtypes of opioid receptors, , ␦, and , are known to form homodimers and heterodimers (10). opioid receptors mediate most of the pain-relieving effects of morphine, the prototypical analgesic used in clinics (11). The observation that antagonism of ␦ receptors or lack of ␦ receptors leads to a reduction in the tolerance that develops on chronic administration of morphine (12) suggests...

Numerous studies have shown that drugs of abuse induce changes in protein expression in the brain that are thought to play a role in synaptic plasticity. Drug-induced plasticity can be mediated by changes at the synapse and more specifically at the postsynaptic density (PSD), which receives and transduces synaptic information. To date, the majority of studies examining synaptic protein profiles have focused on identifying the synaptic proteome. Only a handful of studies have examined the changes in synaptic profile by drug administration. We applied a quantitative proteomics analysis technique with the cleavable ICAT reagent to quantitate relative changes in protein levels of the hippocampal PSD in response to morphine administration. We identified a total of 102 proteins in the mouse hippocampal PSD. The majority of these were signaling, trafficking, and cytoskeletal proteins involved in synaptic plasticity, learning, and memory. Among the proteins whose levels were found to be altered by morphine administration, clathrin levels were increased to the largest extent. Immunoblotting and electron microscopy studies showed that this increase was localized to the PSD. Morphine treatment was also found to lead to a local increase in two other components of the endocytic machinery, dynamin and AP-2, suggesting a critical involvement of the endocytic machinery in the modulatory effects of morphine. Because ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are thought to undergo clathrin-mediated endocytosis, we examined the effect of morphine administration on the association of the AMPA receptor subunit, GluR1, with clathrin. We found a substantial decrease in the levels of GluR1 associated with clathrin. Taken together, these results suggest that, by causing a redistribution of endocytic proteins at the synapse, morphine modulates synaptic plasticity at hippocampal glutamatergic synapses. Molecular & Cellular Proteomics 6:29 -42, 2007.Opiates are choice analgesics in the treatment of chronic pain. However, repeated opiate administration can lead to the development of tolerance, physical dependence, and addiction. Opiate addiction is thought to involve the brain reward circuit as well as brain regions involved in learning and memory, such as the hippocampus (1, 2). There is accumulating evidence that opiates modulate synaptic transmission and plasticity in the hippocampus. For example, opiates have been shown to significantly alter glutamatergic transmission (3), neurogenesis (4), dendritic stability (5), and long term potentiation (6 -8). To date, however, the protein substrates involved in opiate-induced synaptic plasticity in the hippocampus are not well explored.Recent studies have applied genomics and proteomics approaches to identify changes in gene and protein expression in the brain that may be involved in opiate addiction (9 -12). A number of reports have shown that repeated morphine administration alters the expression of proteins involved in receptor endocytosis, neurotransmission, energ...