Hepatic Stellate Cells express RGS5.

<p><b>A</b>. <i>Rgs5^LacZ/LacZ</i> mouse liver with X-gal labeling. RGS5⁺ peri-sinusoidal cells are distributed throughout the liver. RGS5 is also expressed in a subset of SMC of the portal vein (yellow arrows). <b>B–G</b>. Immunofluorescence (IF) for cell-specific markers in the <i>Rgs5^LacZ/LacZ</i> liver. <b>B</b>. SMA and α-β-gal show RGS5 expression in vascular SMCs, as expected (arrows). <b>C</b>. GFAP and α-β-gal IF in <i>Rgs5^LacZ/LacZ</i> mouse liver. β-gal⁺ nuclei are visible within GFAP⁺ astrocyte-like HSC cells, localizing RGS5 expression to HSCs (arrows). <b>D</b>. CRBP1 and α-β-gal IF are co-localized in HSC of <i>Rgs5^LacZ/LacZ</i> liver (arrows). <b>E</b>. VWF (a marker of endothelial cells) and α-β-gal are not co-localized. VWF extends through all sinusoids, while β-gal⁺ cells are sparsely distributed. <b>F</b>. F4/80 (a maker of macrophage/Kupffer cells) and β-gal⁺ cells represent distinct cell populations. <b>G</b>. Confocal image of α-GFAP IF showing co-localization of the nuclear α-β-gal (arrows). <b>H</b>. Confocal image of CD31 (a marker of endothelial cells) and α-β-gal. CD31⁺ cells have β-gal⁻ nuclei, while β-gal⁺ nuclei are not associated with CD31⁺ endothelial cells. All scale bars are 100 µm.</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

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 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

<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⁺ HSCs participate in the response to acute hepatic injury.

<p>Anti-GFAP and anti-β-gal immunofluorescence were used to localize HSCs in acutely injured liver tissue. <b>A–F</b>. Low magnification images. <b>G–I</b>. High magnification of <i>Rgs5^LacZ/LacZ</i><b>A,D</b> Uninjured liver tissue from <i>Rgs5^+/+</i> and <i>Rgs5^LacZ/LacZ</i> mice show sparse HSCs distributed throughout the liver. High magnification in uninjured <i>Rgs5^LacZ/LacZ</i> shows HSCs are GFAP⁺ and β-gal⁺. <b>B,E</b>. At 48 hours post injury, HSCs are concentrated in the necrotic foci surrounding the central veins. β-gal⁺ cells (<b>E,H</b>) are associated with GFAP⁺ cells. <b>C,F</b> At 96 hours post injury, HSCs are tightly clustered at the foci of injury. <b>I</b>. β-gal⁺ cells are GFAP⁺. All scale bars are 100 µm.</p

<i>Rgs5^LacZ/LacZ</i> mice have increased liver injury and HSC activation following acute injury.

<p>Livers from <i>Rgs5^+/+</i> and <i>Rgs5^LacZ/LacZ</i> were collected at 96 hr following a single CCl₄ injection. <b>A</b>. and <b>B</b>. H&E stain of <i>Rgs5^+/+</i> mice recover normally from acute injury. Foci of necrosis are centered on the central veins. <b>B</b>. Hepatocytes appear normal. <b>C</b>. and <b>D</b>. <i>Rgs5^LacZ/LacZ</i> livers have extensive ballooning of hepatocytes throughout the liver. Foci of necrosis are present. <b>D</b>. <i>Rgs5^Lacz/LacZ</i> hepatocytes show cleared cytoplasm and centralized nuclei in hepatocytes distant from necrotic foci. Scale bars are 100 µm. <b>E</b>. Quantification of damaged hepatocyte area reveals a significant increase in ballooning in <i>Rgs5^Lacz/LacZ</i> mice compared to <i>Rgs5^+/+</i> mice (n = 8–10; error bars  = SEM; * = p<0.05) <b>F</b>. mRNA expression of markers of HSC activation (Desmin, PDGFRβ, and ET_B) are elevated in <i>Rgs5^Lacz/LacZ</i> mice compared to <i>Rgs5^+/+</i> mice following acute CCl₄ injury. n = 4–6, * = p<0.05 by Mann-Whitney U test.</p

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

RGS5 expression is up-regulated with HSC activation in chronic liver injury.

<p><i>Rgs5^LacZ/LacZ</i> mice were chronically injected with CCl₄ (or oil), twice weekly for 4 weeks. RNA was isolated from whole liver and analyzed by qPCR for expression of Rgs5 and HSC activation markers. <b>A</b>. Chronic CCl₄ treated <i>Rgs5^LacZ/LacZ</i> dab labeled with anti-SMA shows activated HSCs. <b>B</b>. <i>Rgs5^LacZ/LacZ</i> liver immunolabeled with α-β-gal and α-GFAP antibodies. GFAP⁺ HSCs are visible along fibrotic septa bridging the portal veins. β-gal⁺ HSCs are visible <i>in Rgs5^LacZ/LacZ</i> (<b>B</b>) around the fibrotic septa. Scale bars are 100 µm. <b>C</b>. qPCR of <i>Rgs5^+/+</i> liver RNA shows Rgs5 is up-regulated in chronic CCl₄ injury. Collagen 1α expression is elevated and multiple HSC activation markers (PDGFRα, PDGFRβ, and SMA) are increased relative to oil-injected mice. Data is normalized to oil injected mice. n = 6; error bars  = SEM; * = p<.05.</p

RGS5 expression is up-regulated in acute liver injury.

<p>C57BL/6 mice were injected i.p. with 10 µl/g body weight 10% CCl₄ diluted in olive oil 10% (v/v). Livers were collected at 24, 48, 72 hr post injection. RNA was isolated and expression of HSC activation markers was determined by qPCR. <b>A</b>. RGS5 expression is elevated from 24 to 72 hr post injury, peaking at 48 hr. Expression of <b>B</b>. SMA, <b>C</b>. Desmin, and <b>D</b>. PDGFRβ is up-regulated at 24 and 48 hours post injury. <b>E</b>. PDGFRα and <b>F</b>. Col 1a are up-regulated 48 hours post injury and remain high at 72 hr. Data is normalized to expression in untreated (ut) samples. n = 3–5, error bars  = SEM, * = p<0.05.</p