1. Volume 132, Issue 3, Pages 1088-1103 (March 2007)
Pin1 Interacts With a Specific Serine-Proline Motif of Hepatitis B Virus X-Protein to Enhance Hepatocarcinogenesis 
Roberta Pang, Terence K.W. Lee, Ronnie T.P. Poon, Sheung T. Fan, Kam B. Wong, Yok–Lam Kwong, Eric Tse 
Gastroenterology 
Volume 132, Issue 3, Pages (March 2007)
DOI: /j.gastro
Copyright © 2007 AGA Institute Terms and Conditions

2. Figure 1
Expression of Pin1 and HBx in HCC. (A) Representative microphotographs of Pin1 immunohistochemistry in HBV-related HCCs showing strong cytoplasmic and nuclear staining for Pin1 in tumorous tissue (t), as compared with the weak staining in nontumorous liver tissues (nt) (original magnification, 400×). (B) Consecutive sections of a HCC specimen showing intense staining of Pin1 (right) and HBx in both nucleus and cytoplasm. A representative case with weak cytoplasmic staining of Pin1 and concordantly low HBx immunoreactivity also is shown (original magnification, 400×). (C) Immunostaining of Pin1 and HBx in a representative HCC case related to HCV, and HCC case negative for both HBV and HCV, showing no immunoreactivity for either antigen (original magnification, 400×). (D) Western immunoblotting confirmed the protein expression pattern of HBx and Pin1 in concordance with the immunohistochemical studies of the 3 representative HCC cases.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
Interaction of Pin1 with phosphorylated HBx. (A) GST-Pin1 pull-down of lysate of HeLa cells transfected with GFP-tagged HBx. Upper panel: positive signals with anti-GFP were obtained with GST-Pin1 and GST-Pin1 K63A as baits, but not with GST-vector or GST-Pin1 W34A. Middle panel: confirmation of the presence of input baits with anti-GST before column loading. Lower panel: Confirmation of the presence of HBx in the HeLa cell lysate with anti-GFP. (B) Lysate from HeLa cells transfected with vector control (left) or pFLAG-HBx (right) immunoprecipitated (ip) with anti-Pin1 or normal IgG and immunoblotted (ib) with anti-FLAG (upper panel) or anti-Pin1 (lower panel). (C) HeLa cell lysates immunoprecipitated with anti-FLAG and immunoblotted with anti-Pin1 or anti-FLAG. (D) Lysates from HepG2 cells transfected with vector control or pFLAG-HBx, and immunoblotted with anti-Pin1 or anti-FLAG. (E) GST pull-down of lysate of HeLa cells transiently transfected with GFP-HBx. Treatment with the MEK inhibitor PD98059 (50 μmol/L for 24 hours) resulted in abrogation of interaction with Pin1 (upper panel). The lower panel represents the immunoblot of input lysates with anti-GST before loading into GST columns.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
Pin1 binding to Ser41-Pro motif of HBx. (A) Generation of the 7 HBx mutants. (B) GST-Pin1 pull-down of lysates of HeLa cells transfected with wild-type and mutant HBx. Binding of Pin1 was detected in wild-type (wt) HBx, and 2 HBx mutants (APSP and SASP) with conserved Ser41-Pro motif (lanes 1–3), but not in HBx mutants with disrupted Ser41 or Pro42 (lanes 4–8) (upper panel). The middle and lower panels confirmed the presence of bait and prey proteins.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
Pin1 augmentation of the transactivation activity of HBx. (A) Effect of Pin1 expression on the steady-state level of HBx (wild-type). HepG2 cells were transfected transiently with GFP-HBx and Pin1. Cells were incubated with 20 μg/mL cycloheximide (chx) for the indicated periods, and Western immunoblotting was performed on whole-cell lysates using anti-GFP antibody. GFP-HBx was degraded rapidly, but it was stabilized with ectopic overexpression of Pin1. Actin was used as loading control. (B) Effect of Pin1 expression on the steady-state level of HBx mutants. HepG2 cells were transfected transiently with Pin1-binding and non–Pin1-binding HBx mutants, GFP-HBx APSP, and GFP-HBx APAP, respectively, and treated with 20 μg/mL cycloheximide for various lengths of time as indicated. Western immunoblotting was performed on whole-cell lysates using anti-GFP antibody. In the absence of Pin1 overexpression, both HBx mutants were degraded rapidly. Ectopic Pin1 expression resulted in stabilization of the Pin1-binding HBx APSP mutant in a similar manner as that observed in wild-type HBx WT, but it had no stabilizing effect on the non–Pin1-binding HBx APAP mutant. (C) Decay kinetics of HBx protein (wild-type and mutants) with Pin1 co-expression. Relative HBx protein levels were determined by densitometry and were normalized to actin expression. In the absence of Pin1 overexpression, wild-type HBx and the 2 mutants were degraded rapidly after treatment with cycloheximide. Pin1 overexpression resulted in stabilization of the wild-type HBx and the Pin1-binding HBx APSP mutant, but not the non–Pin1-binding HBx APAP mutant. (D) Effect of HBx expression on the Pin1 level in HepG2 cells. Cells were transfected transiently with Pin1 and GFP-HBx (wild-type and mutants), and total cell lysates were subjected to Western blot analysis with anti-Pin1. Immunoblotting with anti-GFP also was performed to confirm the relative GFP-HBx levels, and actin was used as loading control. (E) Effects of expressions of wild-type (wt) or mutant HBx and Pin1 on the HBx responsive luciferase reporter construct p Filled bars represent a single transfection of HBx (WT or mutant) and grey bars represent transfection of the corresponding HBx with wild-type Pin1. Expression of the empty vector or Pin1 alone did not lead to activation of the reporter. Expression of HBx alone led to a 3–4 times increase in reporter activity, which was increased to about 8 times with co-expression of Pin1, significantly more than that expected from an additive effect of Pin1 and HBx (P < .001). Similar results were obtained with HBx mutants that bound Pin1 (APSP and SASP). In contrast, with HBx mutants that did not bind Pin1 (SPAP, SPSA, APAP, SASA, AAAA), there was no enhancement of reporter activity when Pin1 was co-expressed. Expression of HBx in wild-type and mutant HBx transfectants used was verified by immunoblotting anti-GFP. (F) Effects of expressions of wild-type (wt) or mutant Pin1 and HBx on p Expression of HBx led to a 3–4 times increase in reporter activity as compared with the empty vector control. Expression of Pin1 in the absence of HBx did not increase reporter activity (filled bars). However, co-expression of Pin1 and HBx led to a significant increase in reporter activity (grey bars), as compared with what might be expected with an additive effect of Pin1 and HBx (P < .003). In contrast, co-expression of Pin1 mutants (W34A and K63A) with HBx did not result in any increase in reporter activity (grey bars) as compared with HBx alone. Expression of Pin1 in the 3 Pin1 transfectants (WT, W34A, and K63A) was verified by immunoblotting with anti-Pin1. (G) Effect of Pin1 and HBx on Bcl-XL, c-myc, and NF-κB (p65) mRNA expression. HepG2 cells were transfected transiently with empty vector, GFP-HBx WT, GFP-HBx APSP, and/or Pin1. As assessed by semiquantitative reverse-transcription PCR, co-expression of Pin1 and wild-type HBx or the Pin1-binding HBx APSP mutant led to increased expression of Bcl-XL, c-myc, and NF-κB (p65). (H) Effect of Pin1 and HBx on Bcl-XL, c-myc, and NF-κB (p65) protein expression. The expression of Bcl-XL, c-myc, and NF-κB (p65) was confirmed by Western blot analysis, which showed similar patterns to that observed in G. (I) Effects of expressions of wild-type (wt) or mutant HBx and Pin1 on HepG2 cellular proliferation. Expression of Pin1 or HBx alone led to a 3–4 times increase in cellular proliferation as compared with the empty vector control. Co-expression of Pin1 and wild-type HBx (grey bar) led to an increase of significantly more than what would be expected with a mere additive effect of Pin1 and HBx. A similar result was observed with the HBx mutant APSP, which bound Pin1. In contrast, with the HBx mutant APAP that did not bind Pin1, co-expression with Pin1 only led to an increase in proliferation consistent with the additive effect of Pin1 and HBx. *P < .001 relative to the control group. (J) Effects of expressions of wild-type (wt) or mutant Pin1 and HBx on HepG2 cellular proliferation. Expression of HBx led to a 3–4 times increase in cellular proliferation as compared with the empty vector control. Co-expression of HBx and wild-type Pin1 led to a significant increase in cell proliferation (grey bars), as compared with what might be expected with an additive effect of Pin1 and HBx alone. In contrast, co-expression of both Pin1 mutants (W34A and K63A) with HBx only resulted in an additive effect of Pin1 and HBx. *P < .001 relative to the control group.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 4
Pin1 augmentation of the transactivation activity of HBx. (A) Effect of Pin1 expression on the steady-state level of HBx (wild-type). HepG2 cells were transfected transiently with GFP-HBx and Pin1. Cells were incubated with 20 μg/mL cycloheximide (chx) for the indicated periods, and Western immunoblotting was performed on whole-cell lysates using anti-GFP antibody. GFP-HBx was degraded rapidly, but it was stabilized with ectopic overexpression of Pin1. Actin was used as loading control. (B) Effect of Pin1 expression on the steady-state level of HBx mutants. HepG2 cells were transfected transiently with Pin1-binding and non–Pin1-binding HBx mutants, GFP-HBx APSP, and GFP-HBx APAP, respectively, and treated with 20 μg/mL cycloheximide for various lengths of time as indicated. Western immunoblotting was performed on whole-cell lysates using anti-GFP antibody. In the absence of Pin1 overexpression, both HBx mutants were degraded rapidly. Ectopic Pin1 expression resulted in stabilization of the Pin1-binding HBx APSP mutant in a similar manner as that observed in wild-type HBx WT, but it had no stabilizing effect on the non–Pin1-binding HBx APAP mutant. (C) Decay kinetics of HBx protein (wild-type and mutants) with Pin1 co-expression. Relative HBx protein levels were determined by densitometry and were normalized to actin expression. In the absence of Pin1 overexpression, wild-type HBx and the 2 mutants were degraded rapidly after treatment with cycloheximide. Pin1 overexpression resulted in stabilization of the wild-type HBx and the Pin1-binding HBx APSP mutant, but not the non–Pin1-binding HBx APAP mutant. (D) Effect of HBx expression on the Pin1 level in HepG2 cells. Cells were transfected transiently with Pin1 and GFP-HBx (wild-type and mutants), and total cell lysates were subjected to Western blot analysis with anti-Pin1. Immunoblotting with anti-GFP also was performed to confirm the relative GFP-HBx levels, and actin was used as loading control. (E) Effects of expressions of wild-type (wt) or mutant HBx and Pin1 on the HBx responsive luciferase reporter construct p Filled bars represent a single transfection of HBx (WT or mutant) and grey bars represent transfection of the corresponding HBx with wild-type Pin1. Expression of the empty vector or Pin1 alone did not lead to activation of the reporter. Expression of HBx alone led to a 3–4 times increase in reporter activity, which was increased to about 8 times with co-expression of Pin1, significantly more than that expected from an additive effect of Pin1 and HBx (P < .001). Similar results were obtained with HBx mutants that bound Pin1 (APSP and SASP). In contrast, with HBx mutants that did not bind Pin1 (SPAP, SPSA, APAP, SASA, AAAA), there was no enhancement of reporter activity when Pin1 was co-expressed. Expression of HBx in wild-type and mutant HBx transfectants used was verified by immunoblotting anti-GFP. (F) Effects of expressions of wild-type (wt) or mutant Pin1 and HBx on p Expression of HBx led to a 3–4 times increase in reporter activity as compared with the empty vector control. Expression of Pin1 in the absence of HBx did not increase reporter activity (filled bars). However, co-expression of Pin1 and HBx led to a significant increase in reporter activity (grey bars), as compared with what might be expected with an additive effect of Pin1 and HBx (P < .003). In contrast, co-expression of Pin1 mutants (W34A and K63A) with HBx did not result in any increase in reporter activity (grey bars) as compared with HBx alone. Expression of Pin1 in the 3 Pin1 transfectants (WT, W34A, and K63A) was verified by immunoblotting with anti-Pin1. (G) Effect of Pin1 and HBx on Bcl-XL, c-myc, and NF-κB (p65) mRNA expression. HepG2 cells were transfected transiently with empty vector, GFP-HBx WT, GFP-HBx APSP, and/or Pin1. As assessed by semiquantitative reverse-transcription PCR, co-expression of Pin1 and wild-type HBx or the Pin1-binding HBx APSP mutant led to increased expression of Bcl-XL, c-myc, and NF-κB (p65). (H) Effect of Pin1 and HBx on Bcl-XL, c-myc, and NF-κB (p65) protein expression. The expression of Bcl-XL, c-myc, and NF-κB (p65) was confirmed by Western blot analysis, which showed similar patterns to that observed in G. (I) Effects of expressions of wild-type (wt) or mutant HBx and Pin1 on HepG2 cellular proliferation. Expression of Pin1 or HBx alone led to a 3–4 times increase in cellular proliferation as compared with the empty vector control. Co-expression of Pin1 and wild-type HBx (grey bar) led to an increase of significantly more than what would be expected with a mere additive effect of Pin1 and HBx. A similar result was observed with the HBx mutant APSP, which bound Pin1. In contrast, with the HBx mutant APAP that did not bind Pin1, co-expression with Pin1 only led to an increase in proliferation consistent with the additive effect of Pin1 and HBx. *P < .001 relative to the control group. (J) Effects of expressions of wild-type (wt) or mutant Pin1 and HBx on HepG2 cellular proliferation. Expression of HBx led to a 3–4 times increase in cellular proliferation as compared with the empty vector control. Co-expression of HBx and wild-type Pin1 led to a significant increase in cell proliferation (grey bars), as compared with what might be expected with an additive effect of Pin1 and HBx alone. In contrast, co-expression of both Pin1 mutants (W34A and K63A) with HBx only resulted in an additive effect of Pin1 and HBx. *P < .001 relative to the control group.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 5
Interaction of Pin1 and HBx augmented tumorigenicity in nude mice. (A) Effects of Pin1 and HBx on tumorigenicity of MIHA. MIHA-vector was nontumorigenic. Both MIHA-Pin1 and MIHA-HBx formed tumors, with MIHA-Pin1+HBx forming tumors larger than the additive effects of Pin1 and HBx on MIHA growth, suggesting a synergistic interaction between the 2 proteins. Expression of Pin1 and HBx in each of the transfectants was analyzed by immunoblotting with anti-Pin1 and anti-GFP, respectively. Actin was used as loading control. (B) Representative tumors on day 21. MIHA-Pin1+HBx formed significantly larger tumors than MIHA-Pin1 or MIHA-HBx (P < .001). (C) Effects of HBx mutations on the synergistic interaction of Pin1 with HBx. Without Pin1 overexpression, MIHA-HBx, MIHA-HBxAPSP, and MIHA-HBxAPAP formed tumors of similar sizes. With concomitant Pin1 overexpression, MIHA expressing a HBx mutant APAP that did not bind Pin1 formed tumors larger than those without Pin1 overexpression. However, MIHA expressing HBx or the HBx mutant APSP that bound Pin1 formed even larger tumors, consistent with a synergistic interaction between Pin1 and HBx. Expression of Pin1 and HBx in each of the transfectants was analyzed by immunoblotting with anti-Pin1 and anti-GFP, respectively. Actin was used as loading control. (D) Effects of Pin1 suppression on HBx enhanced tumorigenicity. Without Pin1 suppression (nonsense groups, filled bars), mice inoculated with Hep3B transfectants of HBx, HBxAPSP, and HBxAPAP developed significantly larger tumors in comparison with parental cells (P < .001, <.001, and =.013, respectively), with higher stimulatory effects on tumor growth observed in the HBx and HBxAPSP transfectants. Suppression of Pin1 expression (siPin1 groups, grey bars) led to a significant decrease in tumor growth as compared with the nonsense groups (P < .001), and the differential effect previously observed between HBx mutants binding or not binding Pin1 was abolished. In Pin1-suppressed Hep3B, expression of HBx and HBxAPSP still increased tumor growth, but to a lesser extent compared with the nonsense groups, indicating that HBx had a stimulatory effect on Hep3B tumor growth independent of Pin1.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions