A conserved role for Syntaxin-1 in pre- and post-commissural midline axonal guidance in fly, chick, and mouse.

Axonal growth and guidance rely on correct growth cone responses to guidance cues. Unlike the signaling cascades that link axonal growth to cytoskeletal dynamics, little is known about the crosstalk mechanisms between guidance and membrane dynamics and turnover. Recent studies indicate that whereas axonal attraction requires exocytosis, chemorepulsion relies on endocytosis. Indeed, our own studies have shown that Netrin-1/Deleted in Colorectal Cancer (DCC) signaling triggers exocytosis through the SNARE Syntaxin-1 (STX1). However, limited in vivo evidence is available about the role of SNARE proteins in axonal guidance. To address this issue, here we systematically deleted SNARE genes in three species. We show that loss-of-function of STX1 results in pre- and post-commissural axonal guidance defects in the midline of fly, chick, and mouse embryos. Inactivation of VAMP2, Ti-VAMP, and SNAP25 led to additional abnormalities in axonal guidance. We also confirmed that STX1 loss-of-function results in reduced sensitivity of commissural axons to Slit-2 and Netrin-1. Finally, genetic interaction studies in Drosophila show that STX1 interacts with both the Netrin-1/DCC and Robo/Slit pathways. Our data provide evidence of an evolutionarily conserved role of STX1 and SNARE proteins in midline axonal guidance in vivo, by regulating both pre- and post-commissural guidance mechanisms.

The Dual Function of the Polybasic Juxtamembrane Region of Syntaxin 1A in Clamping Spontaneous Release and Stimulating Ca2+-Triggered Release in Neuroendocrine Cells.

The exact function of the polybasic juxtamembrane region (5RK) of the plasma membrane neuronal SNARE, syntaxin 1A (Syx), in vesicle exocytosis, although widely studied, is currently not clear. Here, we addressed the role of 5RK in Ca2+-triggered release, using our Syx-based intramolecular fluorescence resonance energy transfer (FRET) probe, which previously allowed us to resolve a depolarization-induced Ca2+-dependent close-to-open transition (CDO) of Syx that occurs concomitant with evoked release, both in PC12 cells and hippocampal neurons and was abolished upon charge neutralization of 5RK. First, using dynamic FRET analysis in PC12 cells, we show that CDO occurs following assembly of SNARE complexes that include the vesicular SNARE, synaptobrevin 2, and that the participation of 5RK in CDO goes beyond its participation in the final zippering of the complex, because mutations of residues adjacent to 5RK, believed to be crucial for final zippering, do not abolish this transition. In addition, we show that CDO is contingent on membrane phosphatidylinositol 4,5-bisphosphate (PIP2), which is fundamental for maintaining regulated exocytosis, as depletion of membranal PIP2 abolishes CDO. Prompted by these results, which underscore a potentially significant role of 5RK in exocytosis, we next amperometrically analyzed catecholamine release from PC12 cells, revealing that charge neutralization of 5RK promotes spontaneous and inhibits Ca2+-triggered release events. Namely, 5RK acts as a fusion clamp, making release dependent on stimulation by Ca2+SIGNIFICANCE STATEMENT Syntaxin 1A (Syx) is a central protein component of the SNARE complex, which underlies neurotransmitter release. Although widely studied in relation to its participation in SNARE complex formation and its interaction with phosphoinositides, the function of Syx's polybasic juxtamembrane region (5RK) remains unclear. Previously, we showed that a conformational transition of Syx, related to calcium-triggered release, reported by a Syx-based FRET probe, is abolished upon charge neutralization of 5RK (5RK/A). Here we show that this conformational transition is dependent on phosphatidylinositol 4,5-bisphosphate (PIP2) and is related to SNARE complex formation. Subsequently, we show that the 5RK/A mutation enhances spontaneous release and inhibits calcium-triggered release in neuroendocrine cells, indicating a previously unrecognized role of 5RK in neurotransmitter release.

New Roles of Syntaxin-1A in Insulin Granule Exocytosis and Replenishment.

In type-2 diabetes (T2D), severely reduced islet syntaxin-1A (Syn-1A) levels contribute to insulin secretory deficiency. We generated ß-cell-specific Syn-1A-KO (Syn-1A-ßKO) mice to mimic ß-cell Syn-1A deficiency in T2D. Glucose tolerance tests showed that Syn-1A-ßKO mice exhibited blood glucose elevation corresponding to reduced blood insulin levels. Perifusion of Syn-1A-ßKO islets showed impaired first- and second-phase glucose-stimulated insulin secretion (GSIS) resulting from reduction in readily releasable pool and granule pool refilling. To unequivocally determine the ß-cell exocytotic defects caused by Syn-1A deletion, EM and total internal reflection fluorescence microscopy showed that Syn-1A-KO ß-cells had a severe reduction in the number of secretory granules (SGs) docked onto the plasma membrane (PM) at rest and reduced SG recruitment to the PM after glucose stimulation, the latter indicating defects in replenishment of releasable pools required to sustain second-phase GSIS. Whereas reduced predocked SG fusion accounted for reduced first-phase GSIS, selective reduction of exocytosis of short-dock (but not no-dock) newcomer SGs accounted for the reduced second-phase GSIS. These Syn-1A actions on newcomer SGs were partly mediated by Syn-1A interactions with newcomer SG VAMP8.

HPC-1/syntaxin 1A and syntaxin 1B play distinct roles in neuronal survival.

Two types of syntaxin 1 isoforms, HPC-1/syntaxin 1A (STX1A) and syntaxin 1B (STX1B), are thought to have similar functions in exocytosis of synaptic vesicles. STX1A(-/-) mice which we generated previously develop normally, possibly because of compensation by STX1B. We produced STX1B(-/-) mice using targeted gene disruption and investigated their phenotypes. STX1B(-/-) mice were born alive, but died before postnatal day 14, unlike STX1A(-/-) mice. Morphologically, brain development in STX1B(-/-) mice was impaired. In hippocampal neuronal culture, the cell viability of STX1B(-/-) neurons was lower than that of WT or STX1A(-/-) neurons after 9 days. Interestingly, STX1B(-/-) neurons survived on WT or STX1A(-/-) glial feeder layers as well as WT neurons. However, STX1B(-/-) glial feeder layers were less effective at promoting survival of STX1B(-/-) neurons. Conditioned medium from WT or STX1A(-/-) glial cells had a similar effect on survival, but that from STX1B(-/-) did not promote survival. Furthermore, brain-derived neurotrophic factor (BDNF) or neurotrophin-3 supported survival of STX1B(-/-) neurons. BDNF localization in STX1B(-/-) glial cells was disrupted, and BDNF secretion from STX1B(-/-) glial cells was impaired. These results suggest that STX1A and STX1B may play distinct roles in supporting neuronal survival by glia. Syntaxin 1A (STX1A) and syntaxin 1B (STX1B) are thought to have similar functions as SNARE proteins. However, we found that STX1A and STX1B play distinct roles in neuronal survival using STX1A(-/-) mice and STX1B(-/-) mice. STX1B was important for neuronal survival, possibly by regulating the secretion of neurotrophic factors, such as BDNF, from glial cells.

SNARing platelet granule secretion.

In this issue of Blood, Ye et al and Al Hawas et al clarify the roles of 2 key fusion proteins that regulate the agonist-stimulated release of bioactive factors from platelets, and thereby explain the defective hemostasis in patients with 2 rare genetic diseases.

Syntaxin-11, but not syntaxin-2 or syntaxin-4, is required for platelet secretion.

The platelet release reaction plays a critical role in thrombosis and contributes to the events that follow hemostasis. Previous studies have shown that platelet secretion is mediated by Soluble NSF Attachment Protein Receptor (SNARE) proteins from granule and plasma membranes. The SNAREs form transmembrane complexes that mediate membrane fusion and granule cargo release. Although VAMP-8 (v-SNARE) and SNAP-23 (a t-SNARE class) are important for platelet secretion, the identity of the functional syntaxin (another t-SNARE class) has been controversial. Previous studies using anti-syntaxin Abs in permeabilized platelets have suggested roles for both syntaxin-2 and syntaxin-4. In the present study, we tested these conclusions using platelets from syntaxin-knockout mouse strains and from a Familial Hemophagocytic Lymphohistiocytosis type 4 (FHL4) patient. Platelets from syntaxin-2 and syntaxin-4 single- or double-knockout mice had no secretion defect. Platelets from a FHL4 patient deficient in syntaxin-11 had a robust defect in agonist-induced secretion although their morphology, activation, and cargo levels appeared normal. Semiquantitative Western blotting showed that syntaxin-11 is the more abundant syntaxin in both human and murine platelets. Coimmunoprecipitation experiments showed that syntaxin-11 can form SNARE complexes with both VAMP-8 and SNAP-23. The results of the present study indicate that syntaxin-11, but not syntaxin-2 or syntaxin-4, is required for platelet exocytosis.

Regulation of neuronal M-channel gating in an isoform-specific manner: functional interplay between calmodulin and syntaxin 1A.

Whereas neuronal M-type K(+) channels composed of KCNQ2 and KCNQ3 subunits regulate firing properties of neurons, presynaptic KCNQ2 subunits were demonstrated to regulate neurotransmitter release by directly influencing presynaptic function. Two interaction partners of M-channels, syntaxin 1A and calmodulin, are known to act presynaptically, syntaxin serving as a major protein component of the membrane fusion machinery and calmodulin serving as regulator of several processes related to neurotransmitter release. Notably, both partners specifically modulate KCNQ2 but not KCNQ3 subunits, suggesting selective presynaptic targeting to directly regulate exocytosis without interference in neuronal firing properties. Here, having first demonstrated in Xenopus oocytes, using analysis of single-channel biophysics, that both modulators downregulate the open probability of KCNQ2 but not KCNQ3 homomers, we sought to resolve the channel structural determinants that confer the isoform-specific gating downregulation and to get insights into the molecular events underlying this mechanism. We show, using optical, biochemical, electrophysiological, and molecular biology analyses, the existence of constitutive interactions between the N and C termini in homomeric KCNQ2 and KCNQ3 channels in living cells. Furthermore, rearrangement in the relative orientation of the KCNQ2 termini that accompanies reduction in single-channel open probability is induced by both regulators, strongly suggesting that closer N-C termini proximity underlies gating downregulation. Different structural determinants, identified at the N and C termini of KCNQ3, prevent the effects by syntaxin 1A and calmodulin, respectively. Moreover, we show that the syntaxin 1A and calmodulin effects can be additive or blocked at different concentration ranges of calmodulin, bearing physiological significance with regard to presynaptic exocytosis.

Role of mammalian homologue of Caenorhabditis elegans unc-13-1 (Munc13-1) in the recruitment of newcomer insulin granules in both first and second phases of glucose-stimulated insulin secretion in mouse islets.

AIMS/HYPOTHESIS: We have previously reported that the haplodeficient Munc13-1(+/-) mouse exhibits impaired biphasic glucose-stimulated insulin secretion (GSIS), causing glucose intolerance mimicking type 2 diabetes. Glucagon-like peptide-1 (GLP-1) can bypass these insulin-secretory defects in type 2 diabetes, but the mechanism of exocytotic events mediated by GLP-1 in rescuing insulin secretion is unclear. METHODS: The total internal reflection fluorescence microscopy (TIRFM) technique was used to examine single insulin granule fusion events in mouse islet beta cells. RESULTS: There was no difference in the density of docked granules in the resting state between Munc13-1(+/+) and Munc13-1(+/-) mouse islet beta cells. While exocytosis of previously docked granules in Munc13-1(+/-) beta cells is reduced during high-K(+) stimulation as expected, we now find a reduction in additional exocytosis events that account for the major portion of GSIS, namely two types of newcomer granules, one which has a short docking time (short-dock) and another undergoing no docking before exocytosis (no-dock). As mammalian homologue of Caenorhabditis elegans unc-13-1 (Munc13-1) is a phorbol ester substrate, phorbol ester could partially rescue biphasic GSIS in Munc13-1-deficient beta cells by enhancing recruitment of short-dock newcomer granules for exocytosis. The more effective rescue of biphasic GSIS by GLP-1 than by phorbol was due to increased recruitment of both short-dock and no-dock newcomer granules. CONCLUSIONS/INTERPRETATION: Phorbol ester and GLP-1 potentiation of biphasic GSIS are brought about by recruitment of distinct populations of newcomer granules for exocytosis, which may be mediated by Munc13-1 interaction with syntaxin-SNARE complexes other than that formed by syntaxin-1A.

A role for dynamin in triggering ethanol tolerance.

BACKGROUND: A prevailing hypothesis is that the set of genes that underlie the endophenotypes of alcoholism overlap with those responsible for the addicted state. Functional ethanol tolerance, an endophenotype of alcoholism, is defined as a reduced response to ethanol caused by prior ethanol exposure. The neuronal origins of functional rapid tolerance are thought to be a homeostatic response of the nervous system that counters the effects of the drug. Synaptic proteins that regulate neuronal activity are an important evolutionarily conserved target of ethanol. METHODS: We used mutant analysis in Drosophila to identify synaptic proteins that are important for the acquisition of rapid tolerance to sedation with ethanol. Tolerance was assayed by sedating flies with ethanol vapor and comparing the recovery time of flies after their first sedation and their second sedation. Temperature-sensitive paralytic mutants that alter key facets of synaptic neurotransmission, such as the propagation of action potentials, synaptic vesicle fusion, exocytosis, and endocytosis, were tested for the ability to acquire functional tolerance at both the permissive and restrictive temperatures. RESULTS: The shibire gene encodes Drosophila Dynamin. We tested 2 temperature-sensitive alleles of the gene. The shi(ts1) allele blocked tolerance at both the permissive and restrictive temperatures, while shi(ts2) blocked only at the restrictive temperature. Using the temperature-sensitive property of shi(ts2) , we showed that Dynamin function is required concomitant with exposure to ethanol. A temperature-sensitive allele of the Syntaxin 1A gene, Syx1A(3-69), also blocked the acquisition of ethanol tolerance. CONCLUSIONS: We have shown that shibire and Syntaxin 1A are required for the acquisition of rapid functional tolerance to ethanol. Furthermore, the shibire gene product, Dynamin, appears to be required for an immediate early response to ethanol that triggers a cellular response leading to rapid functional tolerance.

Regulation of voltage-gated calcium channels by synaptic proteins.

Calcium entry through neuronal voltage-gated calcium channels into presynaptic nerve terminal is a key step in synaptic exocytosis. In order to receive the calcium signal and trigger fast, efficient and spatially delimited neurotransmitter release, the vesicle-docking/release machinery must be located near the calcium source. In many cases, this close localization is achieved by a direct interaction of several members of the vesicle release machinery with the calcium channels. In turn, the binding of synaptic proteins to presynaptic calcium channels modulates channel activity to provide fine control over calcium entry, and thus modulates synaptic strength. In this chapter we summarize our present knowledge of the molecular mechanisms by which synaptic proteins regulate presynaptic calcium channel activity.

SNAREing voltage-gated K+ and ATP-sensitive K+ channels: tuning beta-cell excitability with syntaxin-1A and other exocytotic proteins.

The three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, syntaxin, SNAP25 (synaptosome-associated protein of 25 kDa), and synaptobrevin, constitute the minimal machinery for exocytosis in secretory cells such as neurons and neuroendocrine cells by forming a series of complexes prior to and during vesicle fusion. It was subsequently found that these SNARE proteins not only participate in vesicle fusion, but also tether with voltage-dependent Ca(2+) channels to form an excitosome that precisely regulates calcium entry at the site of exocytosis. In pancreatic islet beta-cells, ATP-sensitive K(+) (K(ATP)) channel closure by high ATP concentration leads to membrane depolarization, voltage-dependent Ca(2+) channel opening, and insulin secretion, whereas subsequent opening of voltage-gated K(+) (Kv) channels repolarizes the cell to terminate exocytosis. We have obtained evidence that syntaxin-1A physically interacts with Kv2.1 (the predominant Kv in beta-cells) and the sulfonylurea receptor subunit of beta-cell K(ATP) channel to modify their gating behaviors. A model has proposed that the conformational changes of syntaxin-1A during exocytosis induce distinct functional modulations of K(ATP) and Kv2.1 channels in a manner that optimally regulates cell excitability and insulin secretion. Other proteins involved in exocytosis, such as Munc-13, tomosyn, rab3a-interacting molecule, and guanyl nucleotide exchange factor II, have also been implicated in direct or indirect regulation of beta-cell ion channel activities and excitability. This review discusses this interesting aspect that exocytotic proteins not only promote secretion per se, but also fine-tune beta-cell excitability via modulation of ion channel gating.

VAMP-2, SNAP-25A/B and syntaxin-1 in glutamatergic and GABAergic synapses of the rat cerebellar cortex.

BACKGROUND: The aim of this study was to assess the distribution of key SNARE proteins in glutamatergic and GABAergic synapses of the adult rat cerebellar cortex using light microscopy immunohistochemical techniques. Analysis was made of co-localizations of vGluT-1 and vGluT-2, vesicular transporters of glutamate and markers of glutamatergic synapses, or GAD, the GABA synthetic enzyme and marker of GABAergic synapses, with VAMP-2, SNAP-25A/B and syntaxin-1. RESULTS: The examined SNARE proteins were found to be diffusely expressed in glutamatergic synapses, whereas they were rarely observed in GABAergic synapses. However, among glutamatergic synapses, subpopulations which did not contain VAMP-2, SNAP-25A/B and syntaxin-1 were detected. They included virtually all the synapses established by terminals of climbing fibres (immunoreactive for vGluT-2) and some synapses established by terminals of parallel and mossy fibres (immunoreactive for vGluT-1, and for vGluT-1 and 2, respectively). The only GABA synapses expressing the SNARE proteins studied were the synapses established by axon terminals of basket neurons. CONCLUSION: The present study supplies a detailed morphological description of VAMP-2, SNAP-25A/B and syntaxin-1 in the different types of glutamatergic and GABAergic synapses of the rat cerebellar cortex. The examined SNARE proteins characterize most of glutamatergic synapses and only one type of GABAergic synapses. In the subpopulations of glutamatergic and GABAergic synapses lacking the SNARE protein isoforms examined, alternative mechanisms for regulating trafficking of synaptic vesicles may be hypothesized, possibly mediated by different isoforms or homologous proteins.

Munc18-1 as a key regulator of neurosecretion.

Munc18-1 plays essential roles in neurosecretion by interacting with syntaxin-1 and controlling the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) complex. At least three important functions of Munc18-1 have been proposed: (i) molecular chaperone of syntaxin-1 for appropriate localization and expression of syntaxin-1, (ii) priming/stimulation of the SNARE-mediated membrane fusion, and (iii) docking of large dense-core vesicles to the plasma membrane. Similarly, at least two different binding modes have been proposed for the interaction between Munc18-1 and syntaxin-1: (i) binary binding to a 'closed' conformation of syntaxin-1, and (ii) binding to the N-terminal peptide of syntaxin-1, which is thought to enable an interaction with the quaternary SNARE complex and/or further stabilize the binary interaction between Munc18-1 and closed syntaxin-1. Recent structural analyses have identified critical Munc18-1 residues implicated in these different modes of binding. These have recently been tested functionally in rescue experiments using Munc18-1 null neurons, chromaffin cells and Munc18-1/-2 knockdown PC12 cells, allowing remarkable progress to be made in the structural/functional understanding of Munc18-1. In this review, we summarize these recent advances and attempt to propose an updated model of the pleiotropic functions of Munc18-1 in neuroexocytosis.

G alpha(q)-coupled muscarinic acetylcholine receptors enhance nicotinic acetylcholine receptor signaling in Caenorhabditis elegans mating behavior.

In this study, we address why metabotropic and ionotropic cholinergic signaling pathways are used to facilitate motor behaviors. We demonstrate that a G alpha(q)-coupled muscarinic acetylcholine receptor (mAChR) signaling pathway enhances nicotinic acetylcholine receptor (nAChR) signaling to facilitate the insertion of the Caenorhabditis elegans male copulatory spicules into the hermaphrodite during mating. Previous studies showed that ACh (acetylcholine) activates nAChRs on the spicule protractor muscles to induce the attached spicules to extend from the tail. Using the mAChR agonist Oxo M (oxotremorine M), we identified a GAR-3(mAChR)-G alpha(q) pathway that promotes protractor muscle contraction by upregulating nAChR signaling before mating. GAR-3(mAChR) is expressed in the protractor muscles and in the spicule-associated SPC and PCB cholinergic neurons. However, ablation of these neurons or impairing cholinergic transmission reduces drug-induced spicule protraction, suggesting that drug-stimulated neurons directly activate muscle contraction. Behavioral analysis of gar-3 mutants indicates that, in wild-type males, GAR-3(mAChR) expression in the SPC and PCB neurons is required for the male to sustain rhythmic spicule muscle contractions during attempts to breach the vulva. We propose that the GAR-3(mAChR)/G alpha(q) pathway sensitizes the spicule neurons and muscles before and during mating so that the male can respond to hermaphrodite vulva efficiently.

Syntaxin 1A is required for normal in utero development.

We have generated a syntaxin 1A knockout mouse by deletion of exons 3 through 6 and a concomitant insertion of a stop codon in exon 2. Heterozygous knockout animals were viable with no apparent phenotype. In contrast, the vast majority of homozygous animals died in utero, with embryos examined at day E15 showing a drastic reduction in body size and development when compared to WT and heterozygous littermates. Surprisingly, out of a total of 204 offspring from heterozygous breeding pairs only four homozygous animals were born alive and viable. These animals exhibited reduced body weight, but showed only mild behavioral deficiencies. Taken together, our data indicate that syntaxin 1A is an important regulator of normal in utero development, but may not be essential for normal brain function later in life.

N-type Ca2+ channels are affected by full-length mutant huntingtin expression in a mouse model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the amino-terminal region of the huntingtin (htt) protein. In addition to facilitating neurodegeneration, mutant htt is implicated in HD-related alterations of neurotransmission. Previous data showed that htt can modulate N-type voltage-gated Ca2+ channels (Cav2.2), which are essential for presynaptic neurotransmitter release. Thus, to elucidate the mechanism underlying mutant htt-mediated alterations in neurotransmission, we investigated how Cav2.2 is affected by full-length mutant htt expression in a mouse model of HD (BACHD). Our data indicate that young BACHD mice exhibit increased striatal glutamate release, which is reduced to wild type levels following Cav2.2 block. Cav2.2 Ca2+ current-density and plasma membrane expression are increased in BACHD mice, which could account for increased glutamate release. Moreover, mutant htt affects the interaction between Cav2.2 and 2 major channel regulators, namely syntaxin 1A and Gßγ protein. Notably, 12-month old BACHD mice exhibit decreased Cav2.2 cell surface expression and glutamate release, suggesting that Cav2.2 alterations vary according to disease stage.

The N-terminus of the norepinephrine transporter regulates the magnitude and selectivity of the transporter-associated leak current.

The norepinephrine (NE) transporter (NET) mediates the removal of NE from synaptic spaces and is a major target for antidepressants, amphetamine and cocaine. Previously, we have shown that syntaxin 1A (SYN 1A) supports human NET (hNET) cell surface expression, that hNET/SYN 1A interactions are direct and mediated by the hNET N-terminus, and that the hNET/SYN 1A association limits substrate-induced hNET-associated currents [Sung, U., Apparsundaram, S., Galli, A., Kahlig, K.M., Savchenko, V., Schroeter, S., Quick, M.W., Blakely, R.D., 2003. A regulated interaction of syntaxin 1A with the antidepressant-sensitive norepinephrine transporter establishes catecholamine clearance capacity. J. Neurosci. 23, 1697-1709]. These data raise the possibility that the hNET N-terminus, and potentially its interaction with SYN 1A, might regulate other hNET conductance states, including the hNET-mediated leak current. Importantly for monoamine transporters, the leak conductance has been shown to play a critical role in regulating cell membrane potential and possibly neuronal excitability [Quick, M.W., 2003. Regulating the conducting states of a mammalian serotonin transporter. Neuron 40, 537-549]. Here we demonstrate that deletion of the binding domain for SYN 1A in the NET N-terminus robustly enhances the NET-mediated leak current as well as its selectivity for Cl- permeation under particular intracellular ionic compositions. In addition, we show that the NET N-terminus coordinates the ability of intracellular Na+ and Cl- to regulate the leak conductance. These data suggest that the NET N-terminus regulates and defines the ionic specificity of the NET-mediated leak current.

HIV and HCV Co-Culture Promotes Profibrogenic Gene Expression through an Epimorphin-Mediated ERK Signaling Pathway in Hepatic Stellate Cells.

Accelerated fibrosis in patients co-infected with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) has been a major cause of mortality in the highly active anti-retroviral therapy (HAART) era. However, the role of co-infection in accelerating the progression of liver fibrosis, particularly with regard to the effects of co-infection on hepatic stellate cells (HSCs), remains unclear. We hypothesized that HIV and HCV induce liver fibrosis synergistically by altering the regulation of epimorphin production, and thereby indirectly alter HSC function. Here, we examined the effects of epimorphin on HSC proliferation and invasion, and the changes in fibrogenesis-related gene activity in HSCs (LX2) in the presence of inactivated CXCR4-tropic HIV and HCV (JFH1). The combination of HIV and HCV significantly increased epimorphin expression, which increased the proliferation and invasion capabilities of HSCs. Epimorphin also induced the expression of profibrogenic tissue inhibitor of metalloproteinase 1 (TIMP1) in an extracellular signal-regulated kinase (ERK)-dependent manner. These data indicated that the effects of HIV/HCV co-infection on hepatic fibrosis might be mediated in part by EPM. Strategies to limit the expression of EPM might represent a novel therapeutic approach to prevent the progression of hepatic fibrosis during HIV/HCV co-infection.