The PreS2 activator MHBs(t) of hepatitis B virus activates c-raf-1/Erk2 signaling in transgenic mice

Abstract

The large hepatitis B virus (HBV) surface protein (LHBs) and C-terminally truncated middle size surface proteins (MHBs(t)) form the family of the PreS2 activator proteins of HBV. Their transcriptional activator function is based on the cytoplasmic orientation of the PreS2 domain. MHBs(t) activators are paradigmatic for this class of activators. Here we report that MHBs(t) is protein kinase C (PKC)-dependently phosphorylated at Ser28. The integrity of the phosphorylation site is essential for the activator function. MHBs(t) triggers PKC-dependent activation of c-Raf-1/Erk2 signaling that is a prerequisite for MHBs(t)-dependent activation of AP-1 and NF-kappaB. To analyze the pathophysiological relevance of these data in vivo, transgenic mice were established that produce the PreS2 activator MHBs(t) specifically in the liver. In these mice, a permanent PreS2-dependent specific activation of c-Raf-1/Erk2 signaling was observed, resulting in an increased hepatocyte proliferation rate. In transgenics older than 15 months, an increased incidence of liver tumors occurs. These data suggest that PreS2 activators LHBs and MHBs(t) exert a tumor promoter-like function by activation of key enzymes of proliferation control.

Fig. 1. MHBs^t is a phosphoprotein. (A) Highly purified His₆-MHBs^t76 was derived from Sf9 cells. A 40 µg aliquot was subjected to isoelectric focusing under denaturing conditions using immobiline gel strips covering a range of pH 6–10. The separation in the second dimension was performed using a 15% Laemmli gel. The gel was silver stained. (B) Western blot analysis of highly purified His₆-MHBs^t76 derived from Sf9 cells after two-dimensional separation as described in (A) using a PreS2-specific antiserum (F124). (C) Western blot analysis of highly purifed His₆- MHBs^t76 derived from Sf9 cells using a PreS2-specific antiserum (F124). The sample were subjected to acid phosphatase treatment prior to two-dimensional separation. (D) Autoradiograph of Ni-NTA-precipitated and SDS–PAGE-separated His₆-MHBs^t76 (lane 2), His₆-MHBs^t63 (lane 4), His₆-MHBs (lane 7), His₆-MHBs^t76mutI (lane 3), His₆-MHBs^t76mutII (lane 6), His₆-MHBs^t63mutI (lane 5) and His₆-MHBs^t63mutII (lane 8) derived from cellular lysates of [³²P]orthophosphate-labeled, transfected HepG2 cells. Cells transfected with the cloning vector served as negative control (lane 1). The autoradiograph shows that in contrast to full-length MHBs, the functional MHBs^t activators are phosphoproteins.

Fig. 1. MHBs^t is a phosphoprotein. (A) Highly purified His₆-MHBs^t76 was derived from Sf9 cells. A 40 µg aliquot was subjected to isoelectric focusing under denaturing conditions using immobiline gel strips covering a range of pH 6–10. The separation in the second dimension was performed using a 15% Laemmli gel. The gel was silver stained. (B) Western blot analysis of highly purified His₆-MHBs^t76 derived from Sf9 cells after two-dimensional separation as described in (A) using a PreS2-specific antiserum (F124). (C) Western blot analysis of highly purifed His₆- MHBs^t76 derived from Sf9 cells using a PreS2-specific antiserum (F124). The sample were subjected to acid phosphatase treatment prior to two-dimensional separation. (D) Autoradiograph of Ni-NTA-precipitated and SDS–PAGE-separated His₆-MHBs^t76 (lane 2), His₆-MHBs^t63 (lane 4), His₆-MHBs (lane 7), His₆-MHBs^t76mutI (lane 3), His₆-MHBs^t76mutII (lane 6), His₆-MHBs^t63mutI (lane 5) and His₆-MHBs^t63mutII (lane 8) derived from cellular lysates of [³²P]orthophosphate-labeled, transfected HepG2 cells. Cells transfected with the cloning vector served as negative control (lane 1). The autoradiograph shows that in contrast to full-length MHBs, the functional MHBs^t activators are phosphoproteins.

Fig. 2. MHBs^t is phosphorylated between amino acids 27 and 31. (A) Left: autoradiograph of eubacteria-derived MHBs^t76, incubated with cytoplasm derived from CCL13 cells and [γ-³²P]ATP (lane 1); as negative control, MHBs^t76 was incubated in the absence of lysate, which was replaced by an equal volume of homogenization buffer (lane 2). The molecular weight marker is given on the right. Right: autoradiograph of eubacteria-derived MHBs^t76, incubated with cytoplasm derived from CCL13 cells and [γ-³²P]ATP (lane 3); as negative control, MHBs^t76 was replaced by His₆-grb2 (lane 4). (B) Autoradiograph of in vitro phosphorylated MHBs^t fragments (MHBs^t1-63, MHBs^t1-52, MHBs^t11–76, MHBs^t23–76 and MHBs^t1-52*; here the amphiphatic α-helix between amino acids 41 and 52 was replaced by a β-sheet conformation) isolated from the eubacterial expression system. The experiments were performed in duplicate. In the case of MHBs^t11–76, only one-fifth of the amount of MHBs^t employed in the other reactions was used. (C) Autoradiograph of in vivo phosphorylated and Ni-NTA-agarose-precipitated MHBs^t-specific proteins: lane 1, MHBs^t63 mutIa; lane 2, MHBs^t63mutIb; lane 3, MHBs^t63mutIc; lane 4, MHBs^t63mutId; lane 5, wild-type MHBs^t63; and lane 6, control transfected cells. (D) CAT assay after co-transfection of HepG2 cells with reporter plasmid p3xAP-1-CAT and expression plasmid pKSVMHBs^t76, encoding the wild-type, or plasmids pKSVMHBs^t76mutI, pKSVMHBs^t76mutII, pKSVMHBs^t76mutID28, or cloning vector pKSV10 as negative control. Fold inductions are mean values from three independent transfection experiments, calculated as the ratio of the induced values to the vector control. The standard deviation is shown in the diagram.

Fig. 2. MHBs^t is phosphorylated between amino acids 27 and 31. (A) Left: autoradiograph of eubacteria-derived MHBs^t76, incubated with cytoplasm derived from CCL13 cells and [γ-³²P]ATP (lane 1); as negative control, MHBs^t76 was incubated in the absence of lysate, which was replaced by an equal volume of homogenization buffer (lane 2). The molecular weight marker is given on the right. Right: autoradiograph of eubacteria-derived MHBs^t76, incubated with cytoplasm derived from CCL13 cells and [γ-³²P]ATP (lane 3); as negative control, MHBs^t76 was replaced by His₆-grb2 (lane 4). (B) Autoradiograph of in vitro phosphorylated MHBs^t fragments (MHBs^t1-63, MHBs^t1-52, MHBs^t11–76, MHBs^t23–76 and MHBs^t1-52*; here the amphiphatic α-helix between amino acids 41 and 52 was replaced by a β-sheet conformation) isolated from the eubacterial expression system. The experiments were performed in duplicate. In the case of MHBs^t11–76, only one-fifth of the amount of MHBs^t employed in the other reactions was used. (C) Autoradiograph of in vivo phosphorylated and Ni-NTA-agarose-precipitated MHBs^t-specific proteins: lane 1, MHBs^t63 mutIa; lane 2, MHBs^t63mutIb; lane 3, MHBs^t63mutIc; lane 4, MHBs^t63mutId; lane 5, wild-type MHBs^t63; and lane 6, control transfected cells. (D) CAT assay after co-transfection of HepG2 cells with reporter plasmid p3xAP-1-CAT and expression plasmid pKSVMHBs^t76, encoding the wild-type, or plasmids pKSVMHBs^t76mutI, pKSVMHBs^t76mutII, pKSVMHBs^t76mutID28, or cloning vector pKSV10 as negative control. Fold inductions are mean values from three independent transfection experiments, calculated as the ratio of the induced values to the vector control. The standard deviation is shown in the diagram.

Fig. 3. Functionality of PKC is essential for phosphorylation of MHBs^t. (A) Competitive inhibition of in vitro phosphorylation of MHBs^t using increasing concentrations (2 × 10^–5 M, lane 2, to 2 × 10^–4 M, lane 5) of a PKC-specific peptide. In lanes 4 and 5, the lower band represents the phosphorylated peptide. Lane 1 represents the phosphorylation of MHBs^t63 in the absence of the peptide. (B) In vivo labeling using [³²P]orthophosphate of HepG2 cells producing His₆-MHBs^t76 (lanes 1 and 2) grown in the absence (lane 2) or presence of the PKC inhibitor Goe6976 (lane 1). His₆-MHBs^t76 was enriched by Ni-NTA precipitation.

Fig. 4. PKC is activated in MHBst-producing cells: inhibition abolishes MHBs^t-dependent activation of AP-1 and NF-κB. (A and B) Immunofluorescence of HepG2 cells stably transfected with the expression vector pCMVMHBs^t76 (A). Untransfected cells served as the negative control (B). For detection, a mixture of PKCα- and PKCβ-specific antisera was used. Micrographs were taken at a 400× magnification. (C) CCL13 cells were transfected with the MHBs^t76 expression plasmid pKSVMHBs^t76 or with pKSVMHBst⁷⁶mutI. Transfection with pKSV10 served as the control. The activation was determined by the ratio of membrane-bound to cytosolic PKC activity. In the case of control-transfected cells, this ratio was set arbitrarily as 1. The given factors are mean values of four independent experiments. The standard deviation is shown in the diagram. (D) Transient transfection of HepG2 cells with MHBs^t76 expression plasmid pCMVMHBs^t76 and the reporter constructs p3xAP-1-CAT or p2xNF-κB-CAT. Transfection with pCDNA.3 served as the negative control. Classic PKC isoforms were inhibited by the presence of 10 nM Goe6976. Cells were harvested 40 h after transfection. CAT activity was determined as described in Materials and methods. Fold inductions are mean values from four independent transfection experiments, calculated as the ratio of the induced values to the vector control. The standard deviation is shown in the diagram.

Fig. 4. PKC is activated in MHBst-producing cells: inhibition abolishes MHBs^t-dependent activation of AP-1 and NF-κB. (A and B) Immunofluorescence of HepG2 cells stably transfected with the expression vector pCMVMHBs^t76 (A). Untransfected cells served as the negative control (B). For detection, a mixture of PKCα- and PKCβ-specific antisera was used. Micrographs were taken at a 400× magnification. (C) CCL13 cells were transfected with the MHBs^t76 expression plasmid pKSVMHBs^t76 or with pKSVMHBst⁷⁶mutI. Transfection with pKSV10 served as the control. The activation was determined by the ratio of membrane-bound to cytosolic PKC activity. In the case of control-transfected cells, this ratio was set arbitrarily as 1. The given factors are mean values of four independent experiments. The standard deviation is shown in the diagram. (D) Transient transfection of HepG2 cells with MHBs^t76 expression plasmid pCMVMHBs^t76 and the reporter constructs p3xAP-1-CAT or p2xNF-κB-CAT. Transfection with pCDNA.3 served as the negative control. Classic PKC isoforms were inhibited by the presence of 10 nM Goe6976. Cells were harvested 40 h after transfection. CAT activity was determined as described in Materials and methods. Fold inductions are mean values from four independent transfection experiments, calculated as the ratio of the induced values to the vector control. The standard deviation is shown in the diagram.

Fig. 4. PKC is activated in MHBst-producing cells: inhibition abolishes MHBs^t-dependent activation of AP-1 and NF-κB. (A and B) Immunofluorescence of HepG2 cells stably transfected with the expression vector pCMVMHBs^t76 (A). Untransfected cells served as the negative control (B). For detection, a mixture of PKCα- and PKCβ-specific antisera was used. Micrographs were taken at a 400× magnification. (C) CCL13 cells were transfected with the MHBs^t76 expression plasmid pKSVMHBs^t76 or with pKSVMHBst⁷⁶mutI. Transfection with pKSV10 served as the control. The activation was determined by the ratio of membrane-bound to cytosolic PKC activity. In the case of control-transfected cells, this ratio was set arbitrarily as 1. The given factors are mean values of four independent experiments. The standard deviation is shown in the diagram. (D) Transient transfection of HepG2 cells with MHBs^t76 expression plasmid pCMVMHBs^t76 and the reporter constructs p3xAP-1-CAT or p2xNF-κB-CAT. Transfection with pCDNA.3 served as the negative control. Classic PKC isoforms were inhibited by the presence of 10 nM Goe6976. Cells were harvested 40 h after transfection. CAT activity was determined as described in Materials and methods. Fold inductions are mean values from four independent transfection experiments, calculated as the ratio of the induced values to the vector control. The standard deviation is shown in the diagram.

Fig. 5. MHBs^t induces activation of c-Raf-1and Erk2. (A and B) The activity of c-Raf-1 and Erk2 was determined by immunocomplex assays using recombinant MEK or basic myeloglycoprotein (MBP), respectively, as substrates. HepG2 cells were transfected with pCMVMHBs^t76 or with the phosphorylation-deficient mutant pCMVMHBs^t76mutI. TPA stimulation was used as a positive control. The activity of c-Raf-1 or Erk2 in control-transfected cells was set arbitrarily as 1. The given factors are mean values of three independent experiments. The standard deviation is shown in the diagram.

Fig. 6. Functionality of c-Raf-1 kinase is a prerequisite for MHBs^t-dependent activation of AP-1 and NF-κB. HepG2 cells were transiently transfected with the MHBs^t76 expression plasmid pCMVMHBs^t76, the reporter constructs p3xAP-1-CAT or p2xNF-κB-CAT and a transdominant-negative mutant of Ras (RasN17) or c-Raf-1 (HCR13.1) or with an equal amount of the vector pMNC to equalize the total amount of DNA. Transfection with the cloning vector pCDNA.3 served as negative control. At 36 h after transfection, CAT activity was determined. Fold inductions are mean values from three independent transfection experiments, calculated as the ratio of the induced values to the vector control. The standard deviation is shown in the diagram.

Fig. 7. Liver-specific expression of the transgene encoding MHBs^t76. (A) Schematic structure of the transgene encoding MHBs^t76. To direct the expression to the liver, the albumin promoter that was taken from the plasmid pAlb-PSX (Chisari et al., 1985) was used. To increase the expression level, the β-globin intron, derived from plasmid pSWbglob (Werner et al., 1993), was inserted. The HBV-specific fragment nt3174–3221 was fused to the sequence coding for an N-terminal His₆ tag. The poly(A) site was taken from SV40, subcloned in plasmid pAK2 (Lauer et al., 1992). The HBV-specific fragment nt3174–3221 codes for MHBs^t76. (B) Immunofluorescence microscopy of liver sections derived from 5-month-old female MHBs^t76-producing mice (line 2) (tg). Sections derived from sex-matched wild-type littermates served as control (wt). The PreS2-specific mAb F124 (Neurath et al., 1987) was used for detection of MHBs^t76. The staining was performed using a Cy3-conjugated donkey-derived secondary antibody. The micrographs were taken at a 400× magnification. (C) I: western blot analysis of the eluates from Ni-loaded Hitrap chelating columns. The columns were loaded with lysates derived from the liver or kidney of MHBs^t76-producing transgenic mice or their respective littermates (5-month-old, female mice, line I). Elution was performed by a gradient of increasing imidazole concentration. The PreS2-specific mAb HBV 25-19 (Mimms et al., 1990) was used for detection of MHBs^t76l. II: western blot analysis, using a phosphoserine-specific antiserum (Sigma), of the eluates from Ni-loaded Hitrap chelating columns. The columns were loaded with lysates derived from the liver of wild-type mice (lanes 1 and 3) or MHBs^t76-producing transgenic mice (5-month-old, female mice, line I, lane 2; line II, lane 4). The western blot shows that the mouse-derived MHBs^t76 is a phosphoprotein.

Fig. 7. Liver-specific expression of the transgene encoding MHBs^t76. (A) Schematic structure of the transgene encoding MHBs^t76. To direct the expression to the liver, the albumin promoter that was taken from the plasmid pAlb-PSX (Chisari et al., 1985) was used. To increase the expression level, the β-globin intron, derived from plasmid pSWbglob (Werner et al., 1993), was inserted. The HBV-specific fragment nt3174–3221 was fused to the sequence coding for an N-terminal His₆ tag. The poly(A) site was taken from SV40, subcloned in plasmid pAK2 (Lauer et al., 1992). The HBV-specific fragment nt3174–3221 codes for MHBs^t76. (B) Immunofluorescence microscopy of liver sections derived from 5-month-old female MHBs^t76-producing mice (line 2) (tg). Sections derived from sex-matched wild-type littermates served as control (wt). The PreS2-specific mAb F124 (Neurath et al., 1987) was used for detection of MHBs^t76. The staining was performed using a Cy3-conjugated donkey-derived secondary antibody. The micrographs were taken at a 400× magnification. (C) I: western blot analysis of the eluates from Ni-loaded Hitrap chelating columns. The columns were loaded with lysates derived from the liver or kidney of MHBs^t76-producing transgenic mice or their respective littermates (5-month-old, female mice, line I). Elution was performed by a gradient of increasing imidazole concentration. The PreS2-specific mAb HBV 25-19 (Mimms et al., 1990) was used for detection of MHBs^t76l. II: western blot analysis, using a phosphoserine-specific antiserum (Sigma), of the eluates from Ni-loaded Hitrap chelating columns. The columns were loaded with lysates derived from the liver of wild-type mice (lanes 1 and 3) or MHBs^t76-producing transgenic mice (5-month-old, female mice, line I, lane 2; line II, lane 4). The western blot shows that the mouse-derived MHBs^t76 is a phosphoprotein.

Fig. 7. Liver-specific expression of the transgene encoding MHBs^t76. (A) Schematic structure of the transgene encoding MHBs^t76. To direct the expression to the liver, the albumin promoter that was taken from the plasmid pAlb-PSX (Chisari et al., 1985) was used. To increase the expression level, the β-globin intron, derived from plasmid pSWbglob (Werner et al., 1993), was inserted. The HBV-specific fragment nt3174–3221 was fused to the sequence coding for an N-terminal His₆ tag. The poly(A) site was taken from SV40, subcloned in plasmid pAK2 (Lauer et al., 1992). The HBV-specific fragment nt3174–3221 codes for MHBs^t76. (B) Immunofluorescence microscopy of liver sections derived from 5-month-old female MHBs^t76-producing mice (line 2) (tg). Sections derived from sex-matched wild-type littermates served as control (wt). The PreS2-specific mAb F124 (Neurath et al., 1987) was used for detection of MHBs^t76. The staining was performed using a Cy3-conjugated donkey-derived secondary antibody. The micrographs were taken at a 400× magnification. (C) I: western blot analysis of the eluates from Ni-loaded Hitrap chelating columns. The columns were loaded with lysates derived from the liver or kidney of MHBs^t76-producing transgenic mice or their respective littermates (5-month-old, female mice, line I). Elution was performed by a gradient of increasing imidazole concentration. The PreS2-specific mAb HBV 25-19 (Mimms et al., 1990) was used for detection of MHBs^t76l. II: western blot analysis, using a phosphoserine-specific antiserum (Sigma), of the eluates from Ni-loaded Hitrap chelating columns. The columns were loaded with lysates derived from the liver of wild-type mice (lanes 1 and 3) or MHBs^t76-producing transgenic mice (5-month-old, female mice, line I, lane 2; line II, lane 4). The western blot shows that the mouse-derived MHBs^t76 is a phosphoprotein.

Fig. 7. Liver-specific expression of the transgene encoding MHBs^t76. (A) Schematic structure of the transgene encoding MHBs^t76. To direct the expression to the liver, the albumin promoter that was taken from the plasmid pAlb-PSX (Chisari et al., 1985) was used. To increase the expression level, the β-globin intron, derived from plasmid pSWbglob (Werner et al., 1993), was inserted. The HBV-specific fragment nt3174–3221 was fused to the sequence coding for an N-terminal His₆ tag. The poly(A) site was taken from SV40, subcloned in plasmid pAK2 (Lauer et al., 1992). The HBV-specific fragment nt3174–3221 codes for MHBs^t76. (B) Immunofluorescence microscopy of liver sections derived from 5-month-old female MHBs^t76-producing mice (line 2) (tg). Sections derived from sex-matched wild-type littermates served as control (wt). The PreS2-specific mAb F124 (Neurath et al., 1987) was used for detection of MHBs^t76. The staining was performed using a Cy3-conjugated donkey-derived secondary antibody. The micrographs were taken at a 400× magnification. (C) I: western blot analysis of the eluates from Ni-loaded Hitrap chelating columns. The columns were loaded with lysates derived from the liver or kidney of MHBs^t76-producing transgenic mice or their respective littermates (5-month-old, female mice, line I). Elution was performed by a gradient of increasing imidazole concentration. The PreS2-specific mAb HBV 25-19 (Mimms et al., 1990) was used for detection of MHBs^t76l. II: western blot analysis, using a phosphoserine-specific antiserum (Sigma), of the eluates from Ni-loaded Hitrap chelating columns. The columns were loaded with lysates derived from the liver of wild-type mice (lanes 1 and 3) or MHBs^t76-producing transgenic mice (5-month-old, female mice, line I, lane 2; line II, lane 4). The western blot shows that the mouse-derived MHBs^t76 is a phosphoprotein.

Fig. 8. MHBs^t76 triggers in vivo specific activation of the c-Raf-1/Erk2 signal transduction pathway resulting in an increased proliferation rate of the hepatocytes. (A) Immunocomplex assay of c-Raf-1 activity in liver-derived lysates of MHBs^t76-producing mice (lines 1 and 2), of LHBs-producing transgenic mice, or of the corresponding littermates as negative control. In the case of the lysates derived from LHBs- or MHBs^t76-producing mice, the autoradiograph shows a significant activation of c-Raf-1 as compared with the control. The activation factors (f.a.) are mean values of three independent experiments. Both f.a. and standard deviation (s.d.) are shown at the bottom of the figure. (B) Immunocomplex assay of Erk2 activity in liver-derived lysates of MHBs^t76-producing mice (lines 1 and 2), of LHBs-producing transgenic mice or of the corresponding littermates as negative control. In the case of the lysates derived from LHBs- or MHBs^t76-producing mice, the autoradiograph shows a significant activation of Erk2 as compared with the control. The f.a. are mean values of three independent experiments. Both f.a. and s.d. are shown at the bottom of the figure. (C) Western blot analysis of lysates derived from the liver of the two lines of MHBs^t76-producing transgenic mice (lanes 2 and 3) and of the corresponding littermates (lane 1) using a PCNA-specific antiserum. The western blot shows that in the case of the lysates derived from the MHBs^t76-producing transgenics, an elevated amount of PCNA as compared with the control was detectable. The induction factors (f.i.) are mean values of three independent experiments. Both f.i. and s.d. are shown at the bottom of the figure. (D) Immunofluorescence microscopy of liver cryosections derived from a male 6-month-old MHBs^t76-producing mouse (a and b). A sex- and age-matched wild-type littermate served as control. For detection of PCNA (a and c), a PCNA-specific monoclonal antibody (Santa Cruz) was used and was visualized using a Cy3-conjugated anti-mouse secondary antiserum (Dianova). The nuclei were visualized by DAPI staining (b and d).

Fig. 8. MHBs^t76 triggers in vivo specific activation of the c-Raf-1/Erk2 signal transduction pathway resulting in an increased proliferation rate of the hepatocytes. (A) Immunocomplex assay of c-Raf-1 activity in liver-derived lysates of MHBs^t76-producing mice (lines 1 and 2), of LHBs-producing transgenic mice, or of the corresponding littermates as negative control. In the case of the lysates derived from LHBs- or MHBs^t76-producing mice, the autoradiograph shows a significant activation of c-Raf-1 as compared with the control. The activation factors (f.a.) are mean values of three independent experiments. Both f.a. and standard deviation (s.d.) are shown at the bottom of the figure. (B) Immunocomplex assay of Erk2 activity in liver-derived lysates of MHBs^t76-producing mice (lines 1 and 2), of LHBs-producing transgenic mice or of the corresponding littermates as negative control. In the case of the lysates derived from LHBs- or MHBs^t76-producing mice, the autoradiograph shows a significant activation of Erk2 as compared with the control. The f.a. are mean values of three independent experiments. Both f.a. and s.d. are shown at the bottom of the figure. (C) Western blot analysis of lysates derived from the liver of the two lines of MHBs^t76-producing transgenic mice (lanes 2 and 3) and of the corresponding littermates (lane 1) using a PCNA-specific antiserum. The western blot shows that in the case of the lysates derived from the MHBs^t76-producing transgenics, an elevated amount of PCNA as compared with the control was detectable. The induction factors (f.i.) are mean values of three independent experiments. Both f.i. and s.d. are shown at the bottom of the figure. (D) Immunofluorescence microscopy of liver cryosections derived from a male 6-month-old MHBs^t76-producing mouse (a and b). A sex- and age-matched wild-type littermate served as control. For detection of PCNA (a and c), a PCNA-specific monoclonal antibody (Santa Cruz) was used and was visualized using a Cy3-conjugated anti-mouse secondary antiserum (Dianova). The nuclei were visualized by DAPI staining (b and d).

Fig. 8. MHBs^t76 triggers in vivo specific activation of the c-Raf-1/Erk2 signal transduction pathway resulting in an increased proliferation rate of the hepatocytes. (A) Immunocomplex assay of c-Raf-1 activity in liver-derived lysates of MHBs^t76-producing mice (lines 1 and 2), of LHBs-producing transgenic mice, or of the corresponding littermates as negative control. In the case of the lysates derived from LHBs- or MHBs^t76-producing mice, the autoradiograph shows a significant activation of c-Raf-1 as compared with the control. The activation factors (f.a.) are mean values of three independent experiments. Both f.a. and standard deviation (s.d.) are shown at the bottom of the figure. (B) Immunocomplex assay of Erk2 activity in liver-derived lysates of MHBs^t76-producing mice (lines 1 and 2), of LHBs-producing transgenic mice or of the corresponding littermates as negative control. In the case of the lysates derived from LHBs- or MHBs^t76-producing mice, the autoradiograph shows a significant activation of Erk2 as compared with the control. The f.a. are mean values of three independent experiments. Both f.a. and s.d. are shown at the bottom of the figure. (C) Western blot analysis of lysates derived from the liver of the two lines of MHBs^t76-producing transgenic mice (lanes 2 and 3) and of the corresponding littermates (lane 1) using a PCNA-specific antiserum. The western blot shows that in the case of the lysates derived from the MHBs^t76-producing transgenics, an elevated amount of PCNA as compared with the control was detectable. The induction factors (f.i.) are mean values of three independent experiments. Both f.i. and s.d. are shown at the bottom of the figure. (D) Immunofluorescence microscopy of liver cryosections derived from a male 6-month-old MHBs^t76-producing mouse (a and b). A sex- and age-matched wild-type littermate served as control. For detection of PCNA (a and c), a PCNA-specific monoclonal antibody (Santa Cruz) was used and was visualized using a Cy3-conjugated anti-mouse secondary antiserum (Dianova). The nuclei were visualized by DAPI staining (b and d).

Fig. 8. MHBs^t76 triggers in vivo specific activation of the c-Raf-1/Erk2 signal transduction pathway resulting in an increased proliferation rate of the hepatocytes. (A) Immunocomplex assay of c-Raf-1 activity in liver-derived lysates of MHBs^t76-producing mice (lines 1 and 2), of LHBs-producing transgenic mice, or of the corresponding littermates as negative control. In the case of the lysates derived from LHBs- or MHBs^t76-producing mice, the autoradiograph shows a significant activation of c-Raf-1 as compared with the control. The activation factors (f.a.) are mean values of three independent experiments. Both f.a. and standard deviation (s.d.) are shown at the bottom of the figure. (B) Immunocomplex assay of Erk2 activity in liver-derived lysates of MHBs^t76-producing mice (lines 1 and 2), of LHBs-producing transgenic mice or of the corresponding littermates as negative control. In the case of the lysates derived from LHBs- or MHBs^t76-producing mice, the autoradiograph shows a significant activation of Erk2 as compared with the control. The f.a. are mean values of three independent experiments. Both f.a. and s.d. are shown at the bottom of the figure. (C) Western blot analysis of lysates derived from the liver of the two lines of MHBs^t76-producing transgenic mice (lanes 2 and 3) and of the corresponding littermates (lane 1) using a PCNA-specific antiserum. The western blot shows that in the case of the lysates derived from the MHBs^t76-producing transgenics, an elevated amount of PCNA as compared with the control was detectable. The induction factors (f.i.) are mean values of three independent experiments. Both f.i. and s.d. are shown at the bottom of the figure. (D) Immunofluorescence microscopy of liver cryosections derived from a male 6-month-old MHBs^t76-producing mouse (a and b). A sex- and age-matched wild-type littermate served as control. For detection of PCNA (a and c), a PCNA-specific monoclonal antibody (Santa Cruz) was used and was visualized using a Cy3-conjugated anti-mouse secondary antiserum (Dianova). The nuclei were visualized by DAPI staining (b and d).

Fig. 9. Increased incidence of liver tumors in transgenics producing the activator MHBs^t76. van Gieson-stained (A and C) and HE-stained (B and D) sections of paraffin-embedded liver tissue derived from 15-month-old MHBs^t76-producing mice. These sections encompass healthy tissue and tumor tissue. In (C), infiltration in surrounding liver tissue can be observed in the case of a MHBs^t76 mouse with a primary liver tumor. These photographs were taken at 400× magnification. No significant histological change can be observed in the case of (D), which shows HE-stained liver tissue from a 4-month-old MHBs^t76-producing mouse at 100× magnification.

Fig. 10. Schematic representation of the interference of the PreS2-activators with intracellular signal transduction pathways.