1. Volume 123, Issue 1, Pages 352-364 (July 2002)
Impaired liver regeneration in mice by lipopolysaccharide via TNF-α/kallikrein–mediated activation of latent TGF-β 
Kuniharu Akita, Masataka Okuno, Masamichi Enya, Shoko Imai, Hisataka Moriwaki, Norifumi Kawada, Yasuhiro Suzuki, Soichi Kojima 
Gastroenterology 
Volume 123, Issue 1, Pages (July 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Induction of TGF-β–mediated suppression of hepatic regeneration after PH by preadministration of LPS in mice. (A) Mice were given saline or 500 ng/g body wt LPS 24 hours before either sham operation or 67% PH. Mice were killed at the indicated times, and hepatic PCNA labeling index was determined by counting at least 500 HPCs in each lobe and expressed as a percentage of total HPC numbers counted. Line 1, saline + sham operation; line 2, LPS + sham operation; curve 3, saline + PH; curve 4, LPS + PH. Mean value was calculated from 5 fields per specimen for each experimental mouse, and group means were calculated for 5 mice for each experimental group and expressed as the mean ± SEM (n = 5). An asterisk and a dagger, respectively, indicate significant difference (P < 0.01) from lines 1 and 2 and from curve 4 at the respective time points. (B–E) All animals were killed 48 hours after sham operation or PH. (B) Total RNA was extracted from the liver tissue, and mouse TGF-β1 (upper bands), TGF-β2 (second bands), TGF-β3 (third bands), and GAPDH (bottom bands) mRNA levels were assessed by RT-PCR. (C) TGF-β contents in each liver extract were determined by bioassays and expressed as pmol/mg protein. Values represent the mean ± SD (n = 5). An asterisk indicates significant difference (P < 0.01) obtained by comparisons with the other groups. (D) Liver tissue specimens were stained with antibodies for active TGF-β, synaptophysin, CD31, and CD14. Bar = 20 μm. (E) Some mice were injected twice with 12.5 μg/g body wt anti–TGF-β antibody (Ab) or nonimmune antibody (NI Ab) 2 hours before both LPS administration and PH. Mice were killed 48 hours after PH, and hepatic PCNA labeling index was determined. Values represent the mean ± SEM (n = 5). An asterisk indicates significant difference (P < 0.01) obtained by comparisons with columns 2 and 4. Experiments were repeated 3 times for A and C or twice for B, D, and E, and representative results are shown.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Induction of TNF-α–mediated suppression of hepatic regeneration after PH by preadministration of LPS in mice. (A) Mice were treated as described in the legend to Figure 1. Blood was collected at the indicated times, and serum levels of TNF-α were measured using an enzyme-linked immunosorbent assay kit. Values represent the mean ± SD (n = 5). An asterisk indicates significant difference (P < 0.01) obtained by comparisons with open circle and open triangle at the respective corresponding time. (B) After mice were untreated or treated with 500 ng/g body weight LPS for 24 hours, hepatic levels of CD14 mRNA were estimated by RT-PCR. Representative results from 2 mice are presented for each group (n = 5). (C) Mice were untreated or pretreated with either 50 or 500 ng/g body weight LPS for 24 hours before sham operation or PH and thereafter did not receive or received injections of 5 ng/g body weight TNF-α twice at 2 and 24 hours after PH. In addition, mice were twice injected intravenously (2 hours before both 500 ng/g body wt LPS injection and PH) with 12.5 μg/g body weight anti–TNF-α antibody (Ab). Mice were killed 48 hours after surgery. Hepatic PCNA labeling indexes were measured as before. Values represent the mean ± SEM (n = 5). An asterisk indicates significant difference (P < 0.01) obtained by comparisons with columns 1, 3, 5, and 6. For A–C, experiments were repeated 3 times and representative results with similar results are shown.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Restoration of hepatic regeneration in LPS-injected and partially hepatectomized mice by interference with PLK activity. Five mice in each group were given LPS (500 ng/g body weight) before PH. Mice were injected simultaneously with saline (column 1), 12.5 μg/g body weight anti-PLK antibodies (column 2), 12.5 μg/g body wt nonimmune antibody (NI Ab) (column 3), or 15 μg/g body wt APAA (column 4) before both LPS administration and PH. All animals were killed 48 hours after PH, and liver extracts or sections were prepared. (A) PLK activities in liver extracts were determined using a fluorogenic substrate, Z-Phe-Arg-MCA. The dotted line indicates the hepatic PLK activity in saline-injected and partially hepatectomized mice (n = 5). (B) TGF-β contents in liver extracts were determined by a bioassay. (C) PCNA was stained in the liver specimens, and the indexes were determined. Values represent the mean ± SD (n = 5) for A and B or the mean ± SEM (n = 5) for C. An asterisk indicates significant difference (P < 0.01) obtained by comparisons with saline- and NI Ab-injected mice (samples 1 and 3, respectively). Experiments were repeated twice, and representative results with similar results are shown.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Induction by LPS of the TNF-α–mediated production and activation of latent TGF-β in HSC/KC cocultures. (A) Homotypic and heterotypic cultures of rat HSCs and KCs were incubated for 24 hours with serum-free DMEM containing 0.05% BSA (DMEM-BSA) in the absence (control) or presence of either 45 pmol/L rTNF-α, 500 ng/mL LPS, 45 pmol/L rTNF-α plus 50 μg/mL anti–TGF-β antibody, or 500 ng/mL LPS plus either 50 μg/mL anti–TNF-α antibody or 50 μg/mL anti–TGF-β antibody. Cell lysates were prepared from each culture, and total RNA was extracted. Expressions of TGF-β1 (top bands), TGF-β2 (second bands), TGF-β3 (third bands), and GAPDH (bottom bands) were assessed by RT-PCR. (B) KCs (1 × 107 cells) were incubated for 24 hours in 10-cm dishes with 15 mL DMEM-BSA with or without 500 ng/mL LPS. After cultivation, LPS present in the KC-conditioned medium was removed by extensive dialysis against fresh DMEM. A portion of KC-conditioned medium prepared in the presence of LPS was incubated with 50 μg/mL anti–TNF-α antibody (Ab) for 2 hours. Thereafter, HSC cultures were incubated with either fresh DMEM-BSA or KC-conditioned medium for 24 hours, and both the active (slashed box) and total (open box + slashed box) TGF-β concentrations in each culture medium were determined. (B, inset) Homotypic cultures of either HSCs or KCs were incubated with the indicated concentrations of rTNF-α in DMEM-BSA for 24 hours. Culture media were collected, and both the total (curves 1 and 3) and active (curve 2) TGF-β concentrations were measured. Curves 1 and 2, HSC cultures; curve 3, KC cultures. Values represent the mean ± SD (n = 6). An asterisk indicates significant difference (P < 0.01) obtained by a comparison with the control in each culture (inset) or columns 1 and 2 (B). A dagger in B indicates significant difference (P < 0.01) obtained by a comparison with column 3. A dagger in the inset indicates significant difference (P < 0.01) obtained by a comparison with curve 3. For A and B, representative results from 3 independent experiments with similar results are shown.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Induction of PLK-mediated latent TGF-β activation by TNF-α. (A) Homotypic cultures of HSCs were incubated for 24 hours with DMEM-BSA in the absence (control, column 1) or presence of 45 pmol/L rTNF-α without (column 2) or with 1 μmol/L aprotinin (column 3), 1 μmol/L ϵ-aminocaproic acid (EACA; column 4), 1 μmol/L APAA (column 5), or 25 μg/mL anti-PLK (column 6) or nonimmune (NI) antibodies (Ab) (column 7). Culture media were collected, and the concentrations of active TGF-β in each medium were measured. Values represent the mean ± SD (n = 5). An asterisk indicates significant difference (P < 0.01) obtained by a comparison with the control (column 1). A dagger indicates significant difference (P < 0.01) obtained by comparisons with column 2. (B, upper panel) Recombinant latent TGF-β1 (LTGF-β1; 5 μmol/L) was incubated in a tube for 1 hour at 37°C with PBS-BSA containing 0.1 U/mL purified PLK in the absence or presence of 1 μmol/L APAA. Concentrations of active TGF-β in the solution were measured. Values represent the mean ± SD (n = 3). An asterisk indicates significant difference (P < 0.01) obtained by comparisons with columns 1 and 3. (B, lower panel) Recombinant LAP (70 kilodaltons: 3 μmol/L) was incubated in a tube for 0, 15, and 30 minutes at 37°C with PBS-BSA containing 0.05 U/mL PLK. The degradation of LAP was monitored by silver staining after sodium dodecyl sulfate/polyacrylamide gel electrophoresis with 8% resolving gels under nonreducing conditions. For A and B, representative results from 3 independent experiments with similar results are shown. (C) Homotypic cultures of HSCs in plastic dishes were incubated for 24 hours with DMEM-BSA in the absence or presence of 45 pmol/L rTNF-α. Total RNA was extracted from each HSC culture, and prePLK, uPAR, and GAPDH mRNA levels were assessed by RT-PCR. At the same time, cell extracts were prepared with 0.5% Triton X-100 in 0.1 mol/L Tris, pH 8.0. Proteins (20 μg) in each extract were fractionated through sodium dodecyl sulfate/polyacrylamide gel electrophoresis with 8% resolving gels under nonreducing conditions and transferred onto a nitrocellulose filter; protein levels of prePLK/PLK, uPAR, and GAPDH were assessed by Western blot. (D) Cell-associated PLK activity was assayed using the fluorogenic substrate Z-Phe-Arg-MCA. Values represent the mean ± SD (n = 5). An asterisk indicates significant difference (P < 0.01) obtained by a comparison with the control (left column). (E) HSC cultures were incubated for 8 hours with iodinated PLK (final, 1 μg/mL [11.4 nmol/L]) in the absence (columns 1 and 3) and presence (columns 2 and 4) of 50-fold molar excess of unlabeled PLK followed by chemical cross-linkings. Proteins were extracted from each dish and separated through sodium dodecyl sulfate/polyacrylamide gel electrophoresis as before. Thereafter, the iodinated PLK-binding protein complexes were visualized by autoradiography. Arrow, PLK supposed to bind to uPAR; arrowhead, PLK fragment supposed to bind to uPAR. For C–E, experiments were repeated 3 times and representative results with similar results are shown.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Schematic illustration of PLK-dependent activation of latent TGF-β in the liver initiated by LPS stimulation of KCs. LPS stimulates KCs to secrete TNF-α (1). TNF-α enhances cell surface PLK activity in HSCs (2). PLK provokes the proteolytic activation of latent TGF-β on the HSC surface (3). The resultant active TGF-β suppresses proliferation of HPCs, leading to impaired liver regeneration (4).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions