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A potential role of somatostatin and its receptor SSTR4 in the migration of hepatic oval cells
Youngmi Jung, Seh-Hoon Oh, Donghang Zheng, Thomas D Shupe, Rafal P Witek and Bryon E Petersen
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Figure 1.
HOC induction in the 2AAF/PHx model. (a) Hematoxylin-eosin staining at day 11 liver section of 2AAF/PHx-treated rats (
20, inserted image 40, arrows indicate small HOCs). (b) Immunohistochemical staining for OV6 at day 11 liver section of 2AAF/PHx-treated rats. Brown color indicates OV6-positive cells ( 20). (c) Liver section from the same animal as in (a) and (b), stained by anti-mouse immunoglobulin G serving as a negative control ( 20). (d) Liver section from normal liver tissue ( 20). Data shown represent one of three experiments with similar results.
Figure 2.
Increased SST expression in the HOC induction model. (a) RT-PCR of SST in 2AAF/PHx (D9), Thy-1 sorted HOCs (OC), normal hepatocytes (HEP), and normal liver (NL). GAPDH was used as an internal control. Data shown represent one of three experiments with similar results. (b) Western blot analysis for SST in liver tissue obtained from 2AAF/PHx-treated rats. Protein extracted from the brain was employed as positive control and actin as an internal control. Data shown represent one of three experiments with similar results.
Full figure and legend (46K)
Figure 3.
Immunohistochemical analysis for SST in the HOC induction model. (a) Normal liver, (b) PHx-treated liver (12 h post-PHx), and (c) PHx-treated liver (24 h post-PHx) were stained with SST (Texas red). Representative slides were viewed at 20 magnification. DAPI (blue) was employed for nuclear staining. Double immunofluorescent staining for SST (d; Texas red), CD45 (e; FITC), and merged image (f) of (d) and (e) in 2AAF/PHx-treated rats (day 5, 10). (g–i) Double immunofluorescent staining for SST (g; Texas red), OV6 (h; FITC), and merged image (i) of (g) and (h) in 2AAF/PHx-treated rats (day 13, 10). (j) Magnified image from squares in (f). (k) Magnified image from squares in (i) (colocalized cells appear as yellow to orange). Arrows indicate colocalization with SST and CD45 (j) or OV-6 (k). Original magnifications of (j) and (k) are 10 and 20, respectively. Data shown represent one of three experiments with similar results.
Figure 4.
Potential role of SST as chemoattractant for HOCs. (a) Effects of SST on HOC proliferation. HOCs were incubated in 10% FBS supplemented IMDM medium (
, FBS), or serum-free IMDM medium containing 0.5% BSA with SST at 100 nM (
, SST), or without SST (
, control) for the indicated times. These media were changed every day. Each point represents the mean
s.d., n=3, of independent experiments (*
P<0.001). (b) BrdU incorporation assay. Cells were seeded in a six-well plate (4.5 104 cells/well) and grown in IMDM supplemented with 10%FBS. After 48 h, the medium was replaced with serum-free IMDM for 16 h. The cells were subsequently cultured with 0.5% BSA-containing medium, or medium with addition of 10% FBS or SST at a concentration of 100 nM. After 1 h incubation, 100 
M of BrdU was added. Cells were fixed after 24 h. BrdU-positive cells are presented as percentage of total cell number. Data shown represent one of three experiments with similar results (*
P<0.01). (c) Migration assay using Transwell chamber. HOCs were seeded on the top of the chamber with 1, 10, 100, or 1000 nM of SST protein placed in the bottom chamber or 100 nM of SST protein placed in both upper and lower chamberd. Controls were used with no SST protein in either chamber. Data represent the mean values.d. of three independent experiments. Data were normalized for each independent experiment with respect to control migration (*
P<0.05, **
P<0.01, relative chemotactic index vs control).
Figure 5.
Detection of SSTR4 in HOC-aided liver regeneration. (a) RT-PCR analysis of SSTR subtypes in HOCs, normal hepatocytes (HEP), and normal liver (NL). Asterisks indicate no band for SSTR4 in HEP sample. GAPDH was used as internal control. Data shown represent one of three experiments with similar results. (b) Relative quantification of SSTR4 in HOC induction model using real-time PCR. Data represent the mean values.d. (c) Immunohistochemical staining for SSTR4 at day 9 liver section of HOC induction model (original magnification was taken by using a 40 objective). Anti-goat immunoglobulin (IgG) was employed in staining liver section from the 2AAF/PHx-treated rats, in order to serve as negative control. Data shown represent one of three experiments with similar results.
Figure 6.
Expression of SSTR4 by HOCs. (a) Western blot analysis for SSTR4 in Thy-1 sorted cells from 2AAF/PHx-treated rats and total liver tissue from normal rats. Data shown represent one of three experiments with similar results. (b) Double immunofluorescent staining for SSTR4 (Texas red) and Thy-1 or OV6 (FITC) on cytocentrifuged preparations of Thy-1 sorted cells from 2AAF/PHx-treated rats (magnification of the small images is 40; original magnification of the merge images is 40). In merged image, SSTR4 are shown as yellow to orange. Data shown represent one of three experiments with similar results.
Figure 7.
Effects of SST and SSTR4 on HOC migration and actin rearangement. (a) Stimulation of HOC migration by SST and SSTR4. HOCs were subjected to chemotaxis assays with 100 nM SST (SST) or without SST (Con). Cells were treated with anti-SSTR4 antibody (5 and 10 g/ml) and cultured in 100 nM SST-containing medium for 4 h (5Ab and SST and 10Ab and SST). Addition of anti-SSTR4 antibody significantly reduced the chemotactic response. Data were normalized for each independent experiment with respect to control migration (*
P<0.01, relative chemotactic index vs control). Data represent mean values.d. of three independent experiments. (b) Rearrangement of actin filament in HOCs through SST and SSTR4. HOCs were incubated in IMDM medium containing no addition (control) and 100 nM SST-containing medium for 4 h (SST). Cells were pretreated with anti-SSTR4 antibody and then incubated in 100 nM SST-containing medium for 4 h (anti-SSTR4 Ab and SST) (all images 40). Data shown represent one of three experiments with similar results.
