Expression of GP73, A Resident Golgi Membrane Protein, in Viral and Nonviral Liver Disease

GP73 is a novel type II Golgi membrane protein of unknown function that is expressed in the hepatocytes of patients with adult giant-cell hepatitis (Gene 2000;249:53-65). Its expression pattern in human liver disease and the regulation of its expression in hepatocytes have not been systematically studied. The aims of the present study were to compare GP73 protein levels in viral and nonviral human liver disease and in normal livers, to identify its cellular sources, and to study the regulation of its expression in hepatoma cells in vitro. GP73 protein levels were quantitated in explant livers of patients with well-defined disease etiologies and compared with the levels in normal donor livers. GP73-expressing cells were identified immunohistochemically. GP73 expression in vitro was studied by Western blotting and immunofluorescence microscopy in HepG2 and SK-Hep-1 cells and in the HepG2-derived, hepatitis B virus (HBV)-transfected HepG2215 and HepG2T14.1 cell lines. Whole organ levels of GP73 were low in normal livers. Significant increases were found in liver disease due to viral causes (HBV, HCV) or nonviral causes (alcohol-induced liver disease, autoimmune hepatitis). In normal livers, GP73 was constitutively expressed by biliary epithelial cells but not by hepatocytes. Hepatocyte expression of GP73 was dramatically up-regulated in diseased livers, regardless of the etiology, whereas biliary epithelial cell expression did not change appreciably. GP73 was present at high levels in HepG2215 cells (a cell line that supports active HBV replication), but was absent in HepG2T14.1 cells (an HBV-transfected cell line that does not support HBV replication) and in HBV-free HepG2 cells. In SK-Hep-1 cells, GP73 expression was increased in response to interferon gamma (IFN-y), and inhibited by tumor necrosis factor x (TNF-x). In conclusion, increased expression of GP73 in hepatocytes appears to be a general feature of advanced liver disease, and may be regulated via distinct pathways that involve hepatotropic viruses or cytokines

Cathepsin K-Cre causes unexpected germline deletion of genes in mice.

Osteoclasts are terminally differentiated cells that attach to bone and secrete proteases to degrade the bone matrix. The primary protease responsible for the degradation of the organic component of the bone matrix is Cathepsin K, which was largely thought to be unique to osteoclasts. Given its apparent selective expression in osteoclasts, the Cathepsin K promoter has been engineered to drive the expression of Cre recombinase in mice and has been the most relevant tool for generating osteoclast-specific gene loss. In an effort to understand the role of the ARF tumor suppressor in osteoclasts, we crossed Arf (fl/fl) mice to Ctsk(Cre/+) mice, which unexpectedly resulted in the germline loss of Arf. We subsequently confirmed Cre activity in gametes by generating Ctsk(Cre/+); Rosa(+) mice. These results raise significant concerns regarding in vivo bone phenotypes created using Ctsk(Cre/+) mice and warrant further investigation into the role of Cathepsin K in gametes as well as alternative tools for studying osteoclast-specific gene loss in vivo

Expression of GP73, A Resident Golgi Membrane Protein, in Viral and Nonviral Liver Disease

GP73 is a novel type II Golgi membrane protein of unknown function that is expressed in the hepatocytes of patients with adult giant-cell hepatitis (Gene 2000;249:53-65). Its expression pattern in human liver disease and the regulation of its expression in hepatocytes have not been systematically studied. The aims of the present study were to compare GP73 protein levels in viral and nonviral human liver disease and in normal livers, to identify its cellular sources, and to study the regulation of its expression in hepatoma cells in vitro. GP73 protein levels were quantitated in explant livers of patients with well-defined disease etiologies and compared with the levels in normal donor livers. GP73-expressing cells were identified immunohistochemically. GP73 expression in vitro was studied by Western blotting and immunofluorescence microscopy in HepG2 and SK-Hep-1 cells and in the HepG2-derived, hepatitis B virus (HBV)-transfected HepG2215 and HepG2T14.1 cell lines. Whole organ levels of GP73 were low in normal livers. Significant increases were found in liver disease due to viral causes (HBV, HCV) or nonviral causes (alcohol-induced liver disease, autoimmune hepatitis). In normal livers, GP73 was constitutively expressed by biliary epithelial cells but not by hepatocytes. Hepatocyte expression of GP73 was dramatically up-regulated in diseased livers, regardless of the etiology, whereas biliary epithelial cell expression did not change appreciably. GP73 was present at high levels in HepG2215 cells (a cell line that supports active HBV replication), but was absent in HepG2T14.1 cells (an HBV-transfected cell line that does not support HBV replication) and in HBV-free HepG2 cells. In SK-Hep-1 cells, GP73 expression was increased in response to interferon gamma (IFN-y), and inhibited by tumor necrosis factor x (TNF-x). In conclusion, increased expression of GP73 in hepatocytes appears to be a general feature of advanced liver disease, and may be regulated via distinct pathways that involve hepatotropic viruses or cytokines

Cre expression in <i>Ctsk^Cre/+</i> mouse gametes is verified with <i>Rosa⁺</i> mice.

<p>Reproductive organs of indicated genotypes were analyzed for Cre activity by LacZ staining. (A) In ovaries of <i>Rosa⁺</i>; <i>Ctsk^Cre/+</i> mice, Cre activity was detected in oocytes and cells surrounding the developing oocytes (left panel). Controls were negative for LacZ staining (right panel). Scale bar = 50 µM. (B) In testes of <i>Rosa⁺</i>; <i>Ctsk^Cre/+</i> mice, Cre activity was detected primarily in spermatozoa (left panel). Controls were negative for LacZ staining (right panel). Scale bar = 100 µM.</p

Crossing <i>Arf^fl/fl</i> mice with <i>CtsK^Cre/+</i> mice results in germline <i>Arf</i> loss.

<p>(A) Three sets of primer pairs were designed to detect the presence of floxed <i>exon 1beta</i>, which is unique to <i>Arf.</i> (B) PCR products were not detected upon genotyping new mice for the presence of the 5′ loxP site (far left panel). This cannot be attributed to an absence of DNA (middle left panel). Controls are mice that were never crossed with <i>Ctsk^Cre/+</i> mice. Two sets of primer pairs were used to detect the presence of <i>exon 1beta</i>. Each set detects a product indicating loss of <i>exon 1beta</i> (product size labeled as “KO” in table). (C) Immunofluorescent staining for ARF in testis tissues indicates the loss of <i>exon 1beta</i> at the protein level (scale bar = 100 µM).</p

Crossing <i>Arf^fl/fl</i> mice with <i>CtsK^Cre/+</i> mice results in spontaneous tumor formation.

<p>(A) <i>Ctsk^Cre/+</i>; <i>Arf^fl/fl</i> mice display both fibrosarcomas (top image) and lymphomas with splenomegaly (middle and bottom images). (B) Spontaneous tumor development occurred in all <i>Ctsk^Cre/+</i>; <i>Arf^fl/fl</i> mice.</p

Cre is expressed in the gametes of <i>Ctsk^Cre/+</i> mice.

<p>(A) Real-time PCR was used to quantify the presence of Cathepsin K mRNA (blue) and Cre mRNA (red) in ovary (left) and testis (right) for all indicated genotypes (n = 3). Data are represented as means ± SD. A two-tailed t-test was used to generate indicated p values. (B) Ovaries from WT mice analyzed by IHC for Cathepsin K. Top panels, scale bar = 200 µM. Bottom, left panel shows staining without primary antibody. An adjacent section to the control (bottom, middle) was incubated with primary antibody (scale bar = 100 µM). Red box indicates positively-stained oocyte (bottom, right scale bar = 20 µM).</p