Hepatic stellate cells are the major source of CXCL1, as shown by both quantification of secretion and <i>in situ</i> localization.

<p><b>(A)</b> Quantification of CXCL1 secretion in enriched fractions of hepatocytes, KCs, LSECs and HSCs, freshly isolated and stimulated <i>in vitro</i> with LPS (1 ng/mL LPS, black squares) during 24 hours. Data are representative of three separate experiments with six mice in each group; ^<b>#</b><i>P</i><.05. <b>(B)</b> <i>In-situ</i> localization of CXCL1 in the liver. Immunofluorescent detection for CXCL1 (red) and liver cells nuclei (blue) for nuclei first shows CXCL1 expression in the sinusoids throughout liver parenchyma. <b>(C)</b> Higher resolution shows that CXCL1 (red) is expressed by sub-endothelial cells, which also store retinol droplets in separate compartments, as shown by CRBP1 staining (green). The Cellular Retinol Binding Protein-1 (CRBP-1) is the best marker to detect simultaneously both resting (Glial Fibrillary Acidic Protein, GFAP+) and activated (α-Smooth Muscle Actin, αSMA+) stellate cells <i>in situ</i>. Alexa Fluor-546-CXCL1 (red) staining does not colocalize either with Tie2-GFP in LSECs (green, <i>upper panel</i>), or F4/80 in KCs (blue, <i>middle panel</i>), but with AlexaFluor-488-CRBP1 (green, <i>lower panel</i>), staining both resting and activated HSCs. TOPRO3 was used for nuclei vizualisation.</p

Neutrophils migrate in response to CXCL1 secretion following TLR4 activation in hepatic stellate cells.

<p><b>(A)</b> Schematic representation of the neutrophil chemotaxis assay. <b>(B)</b> Quantification of neutrophil migration in response to secretory WT or TLR4 deficient stellate cells. WT stellate cells were treated (WT HSC + anti CXCL1) or not with anti-CXCL1 antibody. As for internal positive control, the migration of neutrophils towards TLR4 deficient stellate cells supplemented with CXCL1 protein (TLR4 HSC + CXCL1) and with CXCL1 protein only (CXCL1) was quantified in only one experiment. Graphs show three experiments with six mice in each group and statistically significant differences (*<i>P</i><.05) between WT HSCs and TLR4 deficient HSCs, as well as between WT HSCs treated or not with anti-CXCL1, are indicated.</p

Cytokine secretion by hepatocytes, KCs, LSECs and HSCs after isolation from the same liver and in response to low levels of LPS.

<p>Liver cells were freshly isolated on density gradient followed by cell sorting and stimulated with LPS (1ng/mL LPS, black bars or 100ng/mL LPS, hatched bars). Cytokine secretion was measured in the same supernatant with a multiplex assay, run in triplicates. Graphs show three experiments with six mice in each group and statistically significant differences (*<i>P</i><.05) between basal LPS stimulation (1ng/mL) and higher LPS stimulation (100ng/mL) are indicated. Lower panel: bright field images of cells right after isolation (Hepatocytes, LSECs, KCs). Images of HSCs at higher resolution show the retinol droplets at Day 0 and the typical shape of the activated stellate cells after 4 days in culture.</p

Genes down-regulated 2-fold or more in the total liver of TLR4 deficient mice compared to WT mice.

<p>Genes down-regulated 2-fold or more in the total liver of TLR4 deficient mice compared to WT mice.</p

Decrease of CXCL1 message and neutrophil counts in TLR4 deficient liver and after antibiotic treatment.

<p><b>(A)</b> CXCL1 expression in the total liver as analyzed by microarrays. Mean values were obtained from three Genechips for three WT and three TLR4 deficient mice. Statistically significant differences between WT and TLR4 deficient mice are indicated by an asterisk, *<i>P</i><.05, Student <i>t</i> test. (<b>B)</b> CXCL1 expression measured by quantitative RT-PCR. The relative quantity of CXCL1 mRNA in the total liver of WT and TLR4 deficient mice is indicated (*<i>P</i><.01). <b>(C)</b> Relative expression of CXCL1 in the liver from untreated or antibiotic-treated (ABT) WT mice and TLR4 deficient mice; *<i>P</i><.01 <b>(D)</b> Neutrophils counts in the total liver. CD11+ Gr1^high TCR- cells among total live leukocytes isolated from WT and TLR4 deficient liver. In Fig 1B, 1C and 1D, data are representative of five separate experiments with six WT mice (treated or not with antibiotics) and five TLR4 mice; ^<b>#</b><i>P</i><.05; unpaired Mann -Whitney test.</p

Regulator of G-Protein Signaling-5 Is a Marker of Hepatic Stellate Cells and Expression Mediates Response to Liver Injury

<div><p>Liver fibrosis is mediated by hepatic stellate cells (HSCs), which respond to a variety of cytokine and growth factors to moderate the response to injury and create extracellular matrix at the site of injury. G-protein coupled receptor (GPCR)-mediated signaling, via endothelin-1 (ET-1) and angiotensin II (AngII), increases HSC contraction, migration and fibrogenesis. Regulator of G-protein signaling-5 (RGS5), an inhibitor of vasoactive GPCR agonists, functions to control GPCR-mediated contraction and hypertrophy in pericytes and smooth muscle cells (SMCs). Therefore we hypothesized that RGS5 controls GPCR signaling in activated HSCs in the context of liver injury. In this study, we localize RGS5 to the HSCs and demonstrate that <i>Rgs5</i> expression is regulated during carbon tetrachloride (CCl₄)-induced acute and chronic liver injury in <i>Rgs5^LacZ/LacZ</i> reporter mice. Furthermore, CCl₄ treated RGS5-null mice develop increased hepatocyte damage and fibrosis in response to CCl₄ and have increased expression of markers of HSC activation. Knockdown of <i>Rgs5</i> enhances ET-1-mediated signaling in HSCs <i>in vitro</i>. Taken together, we demonstrate that RGS5 is a critical regulator of GPCR signaling in HSCs and regulates HSC activation and fibrogenesis in liver injury.</p></div

RGS5 expression is up-regulated in HCC and liver fibrosis.

<p>Sections of liver from <i>Tsc1^fl/fl;Alb^Cre</i> (<b>A</b>,<b>C</b>) and <i>Pten^fl/fl;Alb^Cre</i> (<b>B</b>,<b>D</b>) mice were stained with Masson's trichrome. <b>A</b>. <i>Tsc1^fl/fl;Alb^Cre</i> non-tumor liver is histologically normal. <b>B</b>. <i>Pten^fl/fl;Alb^Cre</i> non-tumor liver tissue is steatotic and shows collagen deposition in sinusoids (blue). <b>C</b>. <i>Tsc1^fl/fl;Alb^Cre</i> tumor tissue shows disorganized architecture and high levels of collagen deposition. <b>D</b>. <i>Pten^fl/fl;Alb^Cre</i> tumor tissue is glandular in appearance, with robust collagen deposition. Scale bars are 100 µm. <b>E–F</b>. RNA was isolated from wild-type normal tissue and matched tumor and non-tumor tissues of <i>Tsc1^fl/fl;Alb^Cre</i> and <i>Pten^fl/fl;Alb^Cre</i><b>E</b>. <i>Tsc1^fl/fl;Alb^Cre</i> mice have normal RGS5, SMA, and Collagen expression in non-tumor parenchyma, and elevated expression in tumor tissue. <b>F</b>. <i>Pten^fl/fl;Alb^Cre</i> mice have elevated expression of RGS5 and collagen in non-tumor parenchyma and in tumors. Data is normalized to expression in wild-type liver tissue. n = 5, error bars  = SEM, * = p<0.05.</p

Knock-down of RGS5 expression enhances endothelin-1-mediated signaling in LX-2 HSCs.

<p>LX2 cells were treated with <i>Rgs5</i> siRNA or non-specific siRNA for 24 hours, then stimulated with 100 nM ET-1 for the indicated times. Whole cell protein extracts were isolated and analyzed by Western blot. <b>A</b>. A representative immunoblot against pERK1/2 demonstrates increased ET-1-mediated signaling in the absence of RGS5 expression; tERK serves as loading control. <b>B</b>. Quantitation of densitometry of (<b>A</b>) n = 7, error bars  = SEM, * = p<0.05.</p

<i>Rgs5^LacZ/LacZ</i> mice have disrupted hepatocyte morphology after injury.

<p>Acute CCl₄-induced injury in <i>Rgs5^+/+</i> mice (<b>A–C</b>) and <i>Rgs5^LacZ/LacZ</i> (<b>D–F</b>). <b>A,D</b>. Uninjured mice are histologically normal. <b>B</b>,<b>E</b> At 48 hr post CCl₄ injection, foci of necrosis are visible central veins in both <i>Rgs5^+/+</i> and <i>Rgs5^LacZ/LacZ</i> mice. <b>C,F</b>. At 96 hr post injury, clearance of necrotic hepatocytes is underway and infiltrating cells remain at the site of injury. <b>F</b>. In <i>Rgs5^LacZ/LacZ</i> mice, hepatocytes throughout the liver have cleared cytoplasm. Scale bars are 100 µm.</p

RGS5 expression is regulated by profibrotic cytokines in concert with ET_B.

<p>LX2 HSCs were treated with TNFα (5 ng/ml), TGFβ (5 ng/ml), PDGF-BB (10 µM), and ET-1 (100 nm) for 24 hr. RNA was collected for qPCR analysis of RGS5 and ET_B expression. Both RGS5 and ET_B are up-regulated by TNFα stimulation and down-regulated by TGFβ stimulation. RGS5 and ET_B expression are correlated, responding similarly to the same stimuli. n = 3, error bars  = SEM, * = p<0.05.</p