Raphael Rozenfeld

Autophagy Releases Lipid That Promotes Fibrogenesis by Activated Hepatic Stellate Cells in Mice and in Human Tissues

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, Atg7(F/F), and Atg7(F/F)-GFAP-Cre mice, as well as the mouse stellate cell line JS1. Fibrosis was induced in mice using CCl(4) 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 CCl(4) 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.

Receptor heterodimerization leads to a switch in signaling: β-arrestin2-mediated ERK activation by μ-δ opioid receptor heterodimers

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 het‐erodimerization of μOR with δ opioid receptors (δOR) leads to a constitutive recruitment of β‐arrestin2 to the receptor complex resulting in changes in the spatiotemporal regulation of ERK1/2 signaling. The involve‐ment 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 (TippΨ), 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.—Rozenfeld, R., Devi, L. A. Receptor heterodimerization leads to a switch in signaling: β‐ar‐restin2‐mediated ERK activation by μ‐δ opioid receptor heterodimers. FASEB J. 21, 2455–2465 (2007)

Increased abundance of opioid receptor heteromers after chronic morphine administration.

The μ-δ opioid heteromer may be a target for alleviation of chronic pain. Drug-Induced Heteromers Opioid receptors are G protein–coupled receptors and are divided into μ, δ, and κ subtypes. Homomers of μ or δ receptors signal through a Gαi-mediated pathway; however, these receptor subtypes can also form heteromers that signal through Gαz- or β-arrestin2–mediated pathways. Although morphine analgesia is mediated primarily through the μ receptor, δ receptor ligands can potentiate μ receptor–mediated signaling, suggesting that the μ-δ heteromer may also be involved in morphine analgesia. Gupta et al. developed an antibody that selectively recognizes μ-δ heteromers and found that the abundance of the μ-δ heteromer in mice increased with chronic administration of morphine. These increases were detected in regions of the brain that are involved in the modulation of pain transmission. These results suggest that the μ-δ heteromer may be a candidate for therapies to alleviate chronic pain syndromes. 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.

Morphine Administration Alters the Profile of Hippocampal Postsynaptic Density-associated Proteins A Proteomics Study Focusing on Endocytic Proteins

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 α-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.

Receptor heteromerization and drug discovery

G-protein-coupled receptors (GPCRs) are membrane proteins that convert extracellular information into intracellular signals. They are involved in many biological processes and therefore represent powerful targets to modulate physiological and pathological states. Recent studies have demonstrated that GPCR activity is regulated by several mechanisms. Among these, protein-protein interactions (and in particular interactions with other receptors leading to heteromerization) has been shown to have an important role in modulating GPCR function. This has expanded their repertoire of signaling and added a new level of regulation to their physiological roles. Emerging studies provide evidence for tissue-specific and disease-specific receptor heteromerization. This suggests that heteromers represent novel drug targets for the identification of selective compounds with potentially fewer side-effects.

Regulation of CB1 cannabinoid receptor trafficking by the adaptor protein AP-3

Cannabinoid receptor 1 (CB1) 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 intra cellular vesicles. The trafficking events leading to this intracellular localization remain controversial. In this study, we examine the differential trafficking of CB1 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 CB1 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‐3δ knockdown leads to enhanced cell surface localization of CB1 receptors. Finally, we show that CB1 receptors in the late endosomal/lysosomal compartment are associated with heterotrimeric G proteins and mediate signal transduction. These results suggest that intracellular CB1 receptors are functional and that their spatial segregation is likely to significantly affect receptor function.—Rozenfeld, R., Devi, L. A. Regulation of CB1 cannabinoid receptor trafficking by the adaptor protein AP‐3. FASEB J. 22, 2311–2322 (2008)

Cell surface targeting of μ-δ opioid receptor heterodimers by RTP4

μ opioid receptors are G protein–coupled receptors that mediate the pain-relieving effects of clinically used analgesics, such as morphine. Accumulating evidence shows that μ-δ opioid heterodimers have a pharmacologic profile distinct from those of the μ or δ 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 μ-δ 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.

AT1R–CB1R heteromerization reveals a new mechanism for the pathogenic properties of angiotensin II

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 Gαi‐ and Gαq‐coupled receptors is often thought to be oligomerization independent. In this study, we examined the mechanism of signal integration between the Gαi‐coupled type I cannabinoid receptor (CB1R) and the Gαq‐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 heteromer‐selective antibodies, we show that AT1R and CB1R form receptor heteromers. We examined the physiological relevance of this interaction in hepatic stellate cells from ethanol‐administered rats in which CB1R is upregulated. We found a significant upregulation of AT1R–CB1R heteromers and enhancement of angiotensin II‐mediated signalling, as compared with cells from control animals. Moreover, blocking CB1R 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.

Endoplasmic reticulum stress induces fibrogenic activity in hepatic stellate cells through autophagy

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 oxidant 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 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 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 IRE1α 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-opioid interactions during neuropathic pain and analgesia.

Opiates and exogenous cannabinoids, both potent analgesics used for the treatment of patients with neuropathic pain, bind to and activate class A G-protein-coupled receptors (GPCRs). Several lines of evidence have recently suggested that opioid and cannabinoid receptors can functionally interact in the central nervous system (CNS). These interactions may be direct, such as through receptor heteromerization, or indirect, such as through signaling cross-talk that includes agonist-mediated release and/or synthesis of endogenous ligands that can activate downstream receptors. Interactions between opioid and cannabinoid receptors may mediate many of the behavioral phenomena associated with the use of these drugs, including the production of acute antinociception and the development of tolerance and cross-tolerance to the antinociceptive effects of opioid and cannabinoid-specific ligands. This review summarizes behavioral, anatomical, and molecular data characterizing these interactions during the development of neuropathic pain and during antinociceptive treatment with these drugs alone or in combination. These studies are critical for understanding how the receptor systems involved in pain relief are altered during acute or chronic pain, and for designing better antinociceptive drug therapies, such as the combined use of opioid and cannabinoid receptor agonists or selective activation of receptor heteromers, that directly target the altered neurophysiology of patients experiencing pain.