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Am J Physiol Cell Physiol 295: C819-C827, 2008. First published July 23, 2008; doi:10.1152/ajpcell.00366.2007
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VASCULAR BIOLOGY

Protein Never in Mitosis Gene A Interacting-1 (PIN1) regulates degradation of inducible nitric oxide synthase in endothelial cells

Tongzheng Liu, Yongcheng Huang, Rostislav I. Likhotvorik, Lakhu Keshvara, and Dale G. Hoyt

Division of Pharmacology, The Ohio State University College of Pharmacy, and The Dorothy M. Davis Heart and Lung Research Institute, Columbus, Ohio

Submitted 15 August 2007 ; accepted in final form 18 July 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The peptidyl-proline isomerase Protein Never in Mitosis Gene A Interacting-1 (PIN1) increases the level or activity of several transcription factors that can induce the inducible nitric oxide (NO) synthase (iNOS). PIN1 can also regulate mRNA and protein turnover. Here, the effect of depletion of PIN1 on induction of iNOS by Escherichia coli endotoxin (LPS) and interferon-{gamma} (IFN{gamma}) in murine aortic endothelial cells (MAEC) was determined. Suppression of PIN1 by 85% with small hairpin RNA enhanced the induction of NO and iNOS protein by LPS-IFN{gamma}. There was no effect on induction of iNOS mRNA, suggesting a posttranscriptional effect. The enhanced levels of iNOS protein were functionally significant since LPS-IFN{gamma} was cytotoxic to MAEC lacking PIN1 but not MAEC harboring an inactive control construct, and because cytotoxicity was blocked by the NO synthase inhibitor N{omega}-nitro-L-arginine methyl ester. Consistent with posttranscriptional action, knockdown of PIN1 increased the stability of iNOS protein in cycloheximide-treated cells. Furthermore, loss of iNOS was blocked by the calpain inhibitor carbobenzoxy-valinyl-phenylalaninal but not by the selective proteasome inhibitor epoxomicin. Immunoprecipitation indicated that PIN1 can interact with iNOS. Pull down of iNOS with a wild-type glutathione-S-transferase-PIN1 fusion protein, but not with a mutant of the amino terminal phospho-(serine/threonine)-proline binding WW domain of PIN1, indicated that this domain mediates interaction. The results suggest that PIN1 associates with iNOS and can limit its induction by facilitating calpain-mediated degradation in MAEC.

calpain; endothelium; endotoxin; interferon

CIS-TRANS ISOMERIZATION about the bond between an amino acid and proline regulates protein conformation, interactions, and susceptibility to enzymes, including kinases, phosphatases, and proteases. Interconversion between the cis and trans forms is slow, and proline (P), preceded by phosphorylated (p) serine (S) or threonine (T), isomerizes even more slowly than unphosphorylated sequences. Protein Never in Mitosis Gene A Interacting-1 (PIN1) is the only known cis-trans peptidyl-prolyl isomerase that acts on p(S/T)-P (44, 48). PIN1 binds to p(S/T)-P via its amino terminal WW domain. It also catalyzes proline isomerization with its carboxyl terminal catalytic domain (14, 48).

The fundamental effect of PIN1 on protein conformation may account for its diverse roles in cell cycle progression, proliferation, and transcription (22, 35). PIN1 increases the activity of β-catenin, nuclear factor (NF{kappa}B), and activating protein-1 (AP-1). PIN1 also inhibits degradation of β-catenin and NF{kappa}B p65, increasing their levels (28, 34, 35). These transcription factors are associated with induction of inflammatory gene products. PIN1 is also known to regulate degradation of other proteins and mRNA stability (5, 23, 50).

Inducible nitric oxide (NO) synthase (iNOS) is one of the inflammatory genes activated by β-catenin, NF{kappa}B, and AP-1 (3, 17, 25, 29, 47). After induction, iNOS can produce large amounts of NO from arginine in many cell types, including endothelial cells (11, 12, 29). Whether induction of iNOS is beneficial or pathogenic depends on additional factors, such as the concentration of arginine, cofactors, inhibitors, and oxidants (2, 7, 9, 20, 36). A role for PIN1 in the induction of iNOS has not been determined.

Although PIN1 regulates protein conformation and turnover of many of its identified substrates, its role in NO production has not been investigated. Since PIN1 regulates factors that could affect iNOS, the effect of PIN1 knockdown in murine aortic endothelial cells (MAEC) stimulated with Escherichia coli endotoxin (LPS) and interferon-{gamma} (IFN{gamma}) was determined.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Endothelial cell growth supplement, heparin, phenylmethylsulfonyl fluoride, Bradford reagent, E. coli LPS, serotype 0111:B4, N{omega}-nitro-L-arginine methyl ester (L-NAME), and polybrene were purchased from Sigma Chemical (St. Louis, MO). Recombinant mouse IFN{gamma} was from R&D Systems (Minneapolis, MN). Carbobenzoxy-valinyl-phenylalaninal (zVF-CHO), also known as calpain inhibitor III or MDL-28170, epoxomicin, and cycloheximide (CHX) were obtained from Calbiochem (La Jolla, CA). Fetal bovine serum was purchased from Hyclone Laboratories (Logan, UT). Tris-base, ethylenediamine tetraacetic acid, NaCl, Na3VO4, NaF, Tween 20, sodium dodecyl sulfate, ethidium bromide, and agarose were obtained from Fisher Scientific (Fair Lawn, NJ). Triton X-100 was purchased from Pierce (Rockford, IL). HEK293T cells were obtained from American Type Culture Collection (Manassas, VA). Silica columns were purchased from Quiagen (Valencia, CA). Fugene 6 transfection reagent was obtained from Roche (Indianapolis, IN). Glutathione-sepharose was purchased from Amersham Biosciences (Uppsala, Sweden). Quickchange mutagenesis and RNAMaxx in vitro transcription reagents were obtained from Stratagene (La Jolla, CA). Recombinant dicer was from Ambion (Austin, TX). Dulbecco's minimum essential medium, trypsin, isopropyl-β-d-thiogalactopyranoside, TRIzol, Superscript Reverse Transcriptase, Platinum PFX and Taq DNA polymerases, RNAse-free DNAse, deoxynucleotides, recombinant protein G agarose, and plasmid pcDNA 3.1 CTGFP TOPO were purchased from Invitrogen (Carlsbad, CA). Plasmid pGEX4T-3 was from Pharmacia (Piscataway, NJ). Plasmids for lentivirus production were purchased from Addgene (www.addgene.org), where they were deposited by Drs. Robert Weinberg (pCMV-dR8.2 dvpr and pCMV-VSV-G; Whitehead Institute for Biomedical Research) and David Root (pLKO.1; Massachusetts Institute of Technology).

Anti-iNOS antibody was from Transduction Laboratories (Lexington, KY). Anti-PIN1 was purchased from R&D Systems (Minneapolis, MN). Anti-NFKB p65 antibody is from Rockland Immunochemicals (Gilbertsville, PA). Anti-glutathione-S transferase (GST) was prepared as described previously (13). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies were from Jackson Immunoresearch Laboratories (West Grove, PA). Renaissance Enhanced Chemiluminescence Reagent was purchased from New England Nuclear Life Sciences (Boston, MA).

Cells. MAEC were cultured from aortas as described previously (11). The protocol for cell isolation was carried out under approval of the Ohio State University Animal Care and Use Committee.

Short hairpin RNA. The oligomer 5'-CCG GCC GGG TGT ACT ACT TCA ATC ACT CGA GTG ATT GAA GTA GTA CAC CCG GTT TTT G-3' was designed from murine pin1 (Genbank accession NM 023371) to produce a hairpin structure (self-complimentary sequences underlined) with an intervening loop CTCGAG and targeting the 5' underlined sequence in the PIN1 mRNA. This oligomer was annealed to a complimentary oligomer 5'-AAT TCA AAA ACC GGG TGT ACT ACT TCA ATC ACT CGA GTG ATT GAA GTA GTA CAC CCG G-3' to produce AgeI- and EcoRI-compatible overhangs. The annealed DNA was ligated into AgeI- and EcoRI-restricted pLKO.1 vector with T4 ligase, adjacent to a U6 RNA polymerase promoter to drive transcription the shRNA (15), as described by Moffat et al. (27). A mutated sequence was also produced as a control short hairpin RNA (shRNA) (Fig. 1A).


Figure 1
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  		Fig. 1. Suppression of Protein Never in Mitosis Gene A Interacting-1 (PIN1). A: sequences encoding PIN1 knockdown (KD) short hairpin RNA (shRNA) and inactive control shRNA (Control) in the lentiviral system. B: KD or control were used to generate stable murine aortic endothelial cells (MAEC) clones as described in the MATERIALS AND METHODS. Proteins were extracted from KD shRNA, control shRNA, or uninfected MAEC (None), and Western blotted with antibodies specific for PIN1 and {alpha}-tubulin. C: PIN1 was also suppressed using an small inhibitor RNA (siRNA) pool produced by in vitro transcription of long double-stranded RNA and digestion with recombinant Dicer. Representative blots are shown. Densitometric analysis of signal intensities of images was used to measured PIN1 expression. Bars represent means + SE of 3–4 independent cultures of each group. +P < 0.001 for comparison with between KD and control shRNA.

 
Lentiviral particles were produced as described by Stewart et al. (41). In brief, a mixture of 6 µg pLKO.1 vector, 5 µg pCMV-dR8.2 dvpr, and 2 µg pCMV-VSV-G, complexed with Fugene 6 in Dulbecco's minimum essential medium, was added to HEK293T cells in a 75-cm2 flask. Two days after cotransfection, the virus containing supernatant was collected and filtered (0.45 µm) to remove cell debris. The supernatant was stored at –80°C until used to treat MAEC.

Stable PIN1 knockdown (KD) or control shRNA cells were generated using described methods (24). MAEC were cultured for 24 h and then infected with 500 µl of lentiviral supernatants and 8 µg polybrene/ml. After 24 h the viral solution was removed and fresh medium was added. Cells were then grown with 9 µg puromycin/ml. After cell killing and regrowth, cells were transferred to a new flask with puromycin. This selection was repeated four times. The entire process was repeated with two additional MAEC preparations, resulting in three independent stable KD and control shRNA cell cultures.

Small inhibitory RNA. PIN1 small inhibitory RNA (siRNA) was generated by in vitro transcription from a double-stranded DNA template made by reverse transcription-polymerase chain reaction of MAEC RNA with platinum PFX polymerase and primers containing the T7 RNA polymerase promoter sequence at their 5' ends, as described previously (33). PIN1 cDNA was amplified with sense primer 5'-GCG TAA TAC GAC TCA CTA TAG GGA GAA CGA GGA GAA GCT GCC ACC A and antisense primer 5'-GCG TAA TAC GAC TCA CTA TAG GGA GAT CTG TGC GCA GGA TGA TAT G based on Genbank accession number NM 023371 (underlined bases). A control sequence from green fluorescent protein was also produced as described previously (33). After in vitro transcription of the PCR products with T7 RNA polymerase, long double-stranded RNA was digested with recombinant Dicer. The siRNA was purified and MAEC were transfected by electroporation with 150 nM siRNA, as described (33). Treatments of these MAEC were initiated 24 h after transfection.

Treatments. MAEC were cultured in Dulbecco's minimum essential medium-0.5% fetal bovine serum for 18 h and then treated with this medium alone, which contained 0.54 mM L-arginine, or this medium containing 10 µg LPS and 20 ng IFN{gamma} per milliliter, and other agents for various times. Where necessary, protein synthesis was inhibited with 90 µg cycloheximide/ml.

NO accumulation. Medium was collected, and nitrite, reflecting NO, in medium was measured by the Griess reaction, as described previously (11).

Cytotoxicity. To assess cell injury, MAEC were cultured in 96-well plates and treated as described. Cells were rinsed with PBS and fixed with 3% formaldehyde, and adherent cells were stained with 0.5% crystal violet, solubilized, and A590 measured (18).

mRNA levels. Cells were rinsed with phosphate-buffered saline and lysed with TRIzol reagent, and RNA was extracted and recovered by ethanol precipitation. RNA was dissolved in water, and its concentration was determined by absorbance of an aliquot at 260 nm. Three micrograms of RNA were reverse transcribed, and cDNA was subjected to polymerase chain reactions for murine iNOS, VCAM-1, and β-actin as described previously (10, 12). Fifty percent of each reaction was subjected to agarose electrophoresis and ethidium bromide staining. Gels were digitally photographed.

GST-PIN1 fusion proteins. Murine PIN1 was cloned by reverse transcription and polymerase chain reaction with Platinum PFX Polymerase followed by addition of Platinum Taq polymerase to add A to the 3' ends of the expected 504 base reaction product. Sense (5'-GAA GAT GGC GGA CGA GGA GA-3') and antisense (5'-CCT CAT TCT GTG CGC AGG A-3') primers were designed from Genbank accession number NM 023371. A sample of the reaction was incubated with pcDNA 3.1 CTGFP TOPO as instructed by the manufacturer and then used to transform E. coli DH5{alpha}. Colonies were selected and clones confirmed by complete sequencing. The wild-type plasmid was used for polymerase chain reaction mutagenesis. Primers to generate the lysine-64 to alanine (K64A) mutation were 5'-CTC ACA TCT GCT GGT GGC GCA CAG CCA GTC TCG G-3' and 5'-CCG AGA CTG GCT GTG CGC CAC CAG CAG ATG TGA G-3'. Primers to generate the tryptophan-33 to alanine (W33A) mutation were 5'-CAA CGC CAG CCA GGC GGA GCG GCC CAG C and 5'-GCT GGG CCG CTC CGC CTG GCT GGC GTT G-3'. After digestion of the wild-type template with DpnI and bacterial transformation, positive clones were selected and sequenced to confirm mutagenesis.

To produce GST fusions of these PIN1 proteins, wild-type W33A and K64A plasmids were used as templates in polymerase chain reactions with Platinum PFX polymerase, plasmid-specific sense primer, 5'-GAT CGG ATC CAT GGC GGA CGA GGA GAA GC-3' containing a BamHI restriction site, and antisense primer, 5'-GCA TGC CTG CTA TTG TCT-3', located 3' of the PIN1 stop codon in each plasmid. Each reaction product was digested with BamHI and NotI, isolated from an agarose gel, and ligated into pGEX-4T3 with T4 ligase in frame to the carboxyl terminal of the GST sequence. Ligation reactions were used to transform E. coli, and clones were selected and confirmed by sequencing. GST-PIN1 fusion proteins were produced from transformed E. coli BL21 cells induced with isopropyl-β-d-thiogalactopyranoside. Bacteria were sonicated in phosphate-buffered saline and extracted with 1% Triton X100, and debris was removed by centrifugation (16,000 g x 10 min). The extract was incubated with glutathione-sepharose, washed with 0.1 M Tris pH 7 and stored at 4°C (37).

Immunoprecipitation, GST pulldown, and Western blot analysis. Cells were washed with phosphate-buffered saline and lysed and sonicated in lysis buffer containing 1% Triton X-100, 50 mM Tris (pH 7.5), 250 mM NaCl, 5 mM ethylenediamine tetraacetic acid, 4 mM Na3VO4, 20 mM NaF, 1 mM phenylarsine oxide, 30 µg/ml aprotinin, and 30 µg/ml leupeptin. Nuclei were first isolated from some cells by centrifugation, as described previously (12), and treated with lysis buffer before processing for Western blot analysis. Protein concentration was determined by the method of Bradford (1).

For immunoprecipitation, 500 µg of cell lysate protein were incubated with 5 µg anti-PIN1 antibody for 2 h at 4°C with rocking. Recombinant protein G agarose was added, and samples were incubated for 1 h. For GST pulldowns, 500 µg of MAEC lysate protein were precleared by incubation with glutathione-sepharose at 4°C for 2 h. The supernatant was then incubated with the specific glutathione-sepharose-GST-fusion proteins at 4°C for 2 h.

All samples were then centrifuged 16,000 g x 1 min, and the supernatant was discarded. The agarose or sepharose pellets were washed three times with lysis buffer by resuspension and centrifugation.

For Western blot analysis, 12 µg protein were mixed with 20 µl denaturing sample buffer (62.5 mM Tris, pH 6.7, 10% glycerol, 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol, and 0.003% bromphenol blue). Rinsed agarose or sepharose pellets from immunoprecipitation or GST-pulldown were resuspended in 30 or 100 µl denaturing sample buffer, respectively. Samples were heated for 10 min at 95°C (21). Proteins were separated on 4–20% Tris-gylcine polyacrylamide gels and transferred to nitrocellulose.

After transfer, the blots were blocked with 5% nonfat dry milk in Tris-buffered saline buffer and Tween 20 (0.1% Tween 20, 10 mM Tris, pH 7.5, and 150 mM NaCl) and probed with primary antibody. After blots were washed, appropriate horseradish peroxidase-conjugated secondary antibody was added and incubated for 1 h. Proteins were visualized by using enhanced chemiluminescent reagents, captured on X-ray film, and scanned to produce digital images.

Data analysis. Protein levels in Western blot analyses and polymerase chain reaction products in images were quantified with Image J 1.22d (NIH). Data were analyzed by Student's t-test or analysis of variance (ANOVA) with Bonferroni correction for multiple comparisons (40).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PIN1 was reduced more than 85% with the specifically targeted PIN1 knockdown shRNA vector and not with the inactive control shRNA construct (Fig. 1, A and B). PIN1 protein level in control shRNA cells was similar to that in primary MAEC, indicating that the control shRNA was inactive (Fig. 1B). PIN1 was also suppressed by transient transfection with an siRNA pool produced from a 538-bp reverse transcription/polymerase chain reaction product of PIN1 mRNA that was digested with recombinant Dicer, in comparison with control RNA produced from a green fluorescent protein cDNA (Fig. 1C).

Previously, it was found that cotreatment with LPS and IFN{gamma} was required to induce iNOS in MAEC (11, 12). Here, the effect of PIN1 shRNA on iNOS expression was measured in KD and control shRNA cells stimulated with 10 µg LPS and 20 ng IFN{gamma} per ml. Western blot analysis demonstrated that iNOS was induced more in PIN1 KD than in control shRNA cells 24 h after treatment with LPS-IFN{gamma} (Fig. 2A). Similar results were obtained in two other independent pairs of cells selected for KD and control shRNA (data not shown). Suppression of PIN1 by transient transfection with siRNA also increased the induction of iNOS (Fig. 2B). Therefore, induction of iNOS protein was increased by two different methods of PIN1 suppression. Despite the increases in iNOS protein there was no difference in iNOS mRNA between KD and control shRNA cells from 1 to 48 h after treatment with LPS-IFN{gamma} (Fig. 3, A and B). LPS-IFN{gamma} also induced VCAM-1 mRNA as expected, and, as with iNOS, PIN1 depletion did not increase the levels of this message (Fig. 3C). Nuclear NF{kappa}B p65, which may contribute to transcription of both iNOS and VCAM-1 mRNAs, rose slightly more rapidly in KD MAEC when compared with that of control, but the peak level was similar in KD and control shRNA cells (Fig. 3D). These results are consistent with posttranscriptional regulation of iNOS by PIN1 in MAEC.


Figure 2
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  		Fig. 2. Effect of PIN1 knockdown on inducible nitric oxide (NO) synthase (iNOS) protein. KD and control shRNA MAEC (A) or uninfected MAEC electroporated with diced control or PIN1 siRNA (B) were treated with LPS and interferon-{gamma} (IFN{gamma}) for 24 h. Representative Western blots are shown. Bars represent means + SE ratio of iNOS/{alpha}-tubulin from densitometric analysis of three cultures of each group. +P < 0.001 for comparison between KD and control shRNA.

 

Figure 3
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  		Fig. 3. Effect of PIN1 shRNA on iNOS and VCAM-1 mRNA and nuclear factor-{kappa}B (NF{kappa}B). Total RNA or nuclear protein was isolated from LPS-IFN{gamma}-treated cells at the indicated times. mRNAs encoding iNOS, VCAM-1, and β-actin were determined by reverse transcription-polymerase chain reaction and agarose gel electrophoresis. A: representative images of iNOS and β-actin products in ethidium-stained gels. Quantitation of iNOS (B) and VCAM-1 (C) mRNA-to-β-actin signal ratios. Bars represent means + SE ratio of iNOS to β-actin mRNA from densitometric analysis of images from three independent cultures. *P < 0.05 for comparison with 0 h. +P < 0.05 for comparison between KD and control shRNA. Nuclear NF{kappa}B p65 was assessed by Western blotting (D) and image analysis (E).

 
The Griess reaction demonstrated a low basal level of nitrite in medium of all unstimulated MAEC. LPS-IFN{gamma} caused significant elevation of nitrite in the medium in both KD and control shRNA cells after 48 h. However, the nitrite level was significantly higher in KD when compared with that of control shRNA MAEC (Fig. 4A). To determine the functional significance of the enhanced iNOS levels, KD and control shRNA cells were treated with 1 mM L-NAME, a general NOS inhibitor, and with LPS-IFN{gamma}. Cytotoxicity was measured by crystal violet staining of adherent cells remaining in culture after 48 h. LPS-IFN{gamma} caused no significant detachment of MAEC harboring the control construct that does not suppress PIN1 (Fig. 4B). In contrast, LPS-IFN{gamma} caused significant loss of KD cells, which was prevented by L-NAME. Thus depletion of PIN1 enhanced the induction of iNOS by LPS-IFN{gamma} and promoted NO synthase-dependent cytotoxicity.


Figure 4
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  		Fig. 4. Effect of PIN1 shRNA on NO accumulation and cytotoxicity of LPS and IFN{gamma}. A: MAEC were incubated in Dulbecco's minimum essential medium-0.5% fetal bovine serum overnight and treated with vehicle or 10 µg LPS/ml and 20 ng IFN{gamma}/ml (L/I) for 48 h. The concentration of nitrite in cell culture supernatant was measured by the Griess reaction. Bars represent means + SE of 7 separate determinations. *P < 0.01 for comparison between vehicle and L/I-treated cells. +P < 0.05 for comparison between KD and control shRNA cells. B: PIN1 KD and control shRNA MAEC were pretreated without or with 1 mM N{omega}-nitro-L-arginine methyl ester (L-NAME) for 1 h and then treated vehicle or LPS-IFN{gamma} for 48 h. After crystal violet staining was completed, the percent viability, with vehicle set to 100%, was determined. Bars represent means + SE of of 6–8 separate determinations. *P < 0.0001 for comparison between vehicle and L/I-treated cells. +P < 0.0001 for comparison between KD and control shRNA cells.

 
Since PIN1 knockdown did not affect the induction of iNOS mRNA, degradation of iNOS was investigated. KD and control shRNA cells were treated with 90 µg CHX/ml 24 h after induction with LPS-IFN{gamma} to block translation. Proteins were extracted 0, 8, and 24 h after the addition of CHX and subjected to Western blot analysis. The level of iNOS fell to less than 50% of the initial value by 8 h after CHX in the control shRNA cells. In KD cells, a similar decrease in iNOS was delayed until 24 h after CHX treatment (Fig. 5). The half-life of iNOS protein was ~7 h in control shRNA cells, but it was prolonged to about 24 h in PIN1 KD MAEC.


Figure 5
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  		Fig. 5. PIN1 regulates iNOS protein stability. KD and control shRNA MAEC were treated with LPS and IFN{gamma} for 24 h. The protein synthesis inhibitor cycloheximide (CHX) 90 µg/ml was added, and cell extracts were collected at the indicated times. A: iNOS and {alpha}-tubulin were measured by Western blot analysis. Representative blots are shown. B: iNOS and {alpha}-tubulin levels in each sample were determined by image analysis of Western blots. Ratios of iNOS to {alpha}-tubulin were determined, and the average ratio ± SE of 4 independent cultures for each point, as a percentage of the value at 0 h after CHX treatment, was calculated. The dashed line marks the 50% value. +P < 0.05 for comparison between KD and control shRNA at the indicated time.

 
Others found that calpain degraded iNOS in RAW 264.7 cells and in vitro (43). Here, KD and control shRNA MAEC were treated with vehicle or the calpain inhibitor zVF-CHO (25 µM) for 1 h and then stimulated with LPS-IFN{gamma} for 24 h. zVF-CHO significantly increased induction of iNOS by LPS-IFN{gamma} in KD and control shRNA cells. Induction of iNOS was again greater in KD when compared with control shRNA cells (Fig. 6, A and B). The effect of zVF-CHO on degradation of iNOS was next examined by treating control shRNA MAEC with CHX 24 h after induction with LPS-IFN{gamma}. The drop in iNOS seen after 8 h in vehicle-treated cells was prevented by zVF-CHO. The value only decreased to 59% of the starting value 24 h after CHX in cells exposed to zVF-CHO compared with 14% in cells incubated without the calpain inhibitor (Fig. 6C).


Figure 6
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  		Fig. 6. Effect of calpain inhibition on iNOS. A: PIN1 KD and control shRNA MAEC were pretreated with vehicle or zVF-CHO (25 µM) for 1 h and then treated with LPS and IFN{gamma} for 24 h. Representative Western blot analyses of iNOS are shown. B: densitometric analysis of signal intensities of images from three samples per group for the ratio of iNOS to {alpha}-tubulin. Bars represent the means + SE of three independent cultures per group. *P < 0.05 for comparison between vehicle and zVF-CHO. +P < 0.05 for comparison between KD and control shRNA. C: control shRNA MAEC were treated as in A and then treated with CHX (90 µg/ml). Cell extracts were collected at the indicated times after CHX and analyzed by Western blot analysis for iNOS and {alpha}-tubulin. Ratios of iNOS to {alpha}-tubulin were determined. The mean percentage of the value at 0 h after CHX treatment ± SE of 2–4 independent cultures per group is shown. The dashed line marks the 50% value. +P = 0.018 and *P = 0.066 for one-tail comparison between vehicle and zVF-CHO at the indicated time.

 
The ubiquitin-proteasome pathway can target iNOS for degradation, and it affects iNOS transcription by degrading inhibitor of {kappa}B (I{kappa}B) {alpha}/β. The latter effect increases levels of active NF{kappa}B, which can induce iNOS mRNA (30). When KD and control shRNA MAEC were initially treated with 2 µM epoxomicin, a selective proteasome inhibitor (26), for 1 h and then stimulated with LPS-IFN{gamma}, induction of iNOS was prevented (Fig. 7A). As expected the blockage of iNOS induction by epoxomicin was associated with increased levels of I{kappa}B{alpha} and β in LPS-IFN{gamma}-treated cells (Fig. 7B). I{kappa}B{alpha}/β levels were similar in KD and control shRNA MAEC. MAEC were then treated with LPS-IFN{gamma} for 24 h to induce iNOS, followed with epoxomicin and 90 µg CHX/ml for 0, 8, or 24 h. Epoxomicin had no effect on the loss of the induced iNOS protein over time after CHX in control shRNA MAEC (Fig. 7C).


Figure 7
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  		Fig. 7. Effect of proteasome inhibition on iNOS. A: MAEC were pretreated with vehicle or epoxomicin (EPO, 2 µM) for 1 h and then treated with LPS and IFN{gamma} for 24 h. Representative Western blot analyses of iNOS and {alpha}-tubulin in KD and control shRNA. B: MAEC were pretreated with vehicle or epoxomicin for 1 h and then without or with LPS and IFN{gamma} (L/I) for 15 min. Extracts were analyzed by Western blot analysis using anti-I{kappa}B-{alpha}/β antibody. C: control shRNA MAEC were treated LPS and IFN{gamma} for 24 h, then with epoxomicin for 1 h, and finally with CHX (90 µg/ml). Cell extracts were collected at the indicated times after CHX and analyzed by Western blot analysis for iNOS and {alpha}-tubulin. Ratios of iNOS to {alpha}-tubulin were determined. The mean percentage of the value at 0 h after CHX treatment ± SE of 2–4 independent cultures per group is shown.

 
The potential for interaction between iNOS and PIN1 was assessed. iNOS was coimmunoprecipitated with PIN1 in control shRNA MAEC treated with LPS-IFN{gamma} (Fig. 8A).


Figure 8
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  		Fig. 8. PIN1 interacts with iNOS. A: control shRNA MAEC were treated with LPS-IFN{gamma} for 24 h. Cell extracts were immunoprecipitated without (None) or with anti-PIN1 antibody (PIN1) and analyzed by Western blot analysis with anti-iNOS antibody. B: cell extracts were incubated with GST or GST-wild-type PIN1, -W33A or -K64A fusion proteins, and glutathione sepharose, and analyzed by Western blotting with anti-iNOS and anti-GST antibodies. Blots are representative of 3 independent cultures.

 
To investigate requirements for PIN1 domains in interaction with iNOS, a WW domain mutant, W33A, which does not bind p(S/T)-P sites in substrates, and a peptidyl-prolyl isomerase catalytic mutant, K64A, were generated. Samples were incubated with glutathione-sepharose and GST, GST-PIN1, GST-W33A PIN1, or GST-K64A PIN1. GST failed to bind iNOS, but iNOS was pulled down by GST-PIN1. GST-W33A PIN1 interacted poorly with iNOS, whereas the K64A catalytic mutant pulled down a detectable level of iNOS (Fig. 8B).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The role of PIN1 in the inflammatory response of endothelial cells has not been described. PIN1 is known to increase the level and/or activity of several transcription factors that mediate induction of iNOS (3, 12, 28, 35, 45, 47). It was expected that suppression of PIN1 would limit the induction of iNOS by LPS-IFN{gamma} in MAEC. However, depletion of PIN1 by 85% increased induction of iNOS protein, whereas increases in iNOS mRNA caused by LPS-IFN{gamma} were not affected (Fig. 3, A and B). Experiments in cytokine-treated PIN1 knockout murine embryonic fibroblasts and in unstimulated siRNA-treated cancer cells, indicated that NF{kappa}B activation of a reporter gene was reduced. In cytokine-stimulated knockout embryonic fibroblasts, NF{kappa}B activity was impaired, probably as a result of increased proteasomal degradation of p65 in the complete absence of PIN1 (35). Partial suppression of PIN1 in MAEC here did not affect the peak level of nuclear NF{kappa}B p65, induction of endogenous iNOS mRNA, or of another p65-regulated mRNA, VCAM-1, in MAEC (Fig. 3). We speculate that different transcription factors and proteins may be significantly affected at different levels of PIN1, and the level of PIN1 required for expression, function, and turnover of different proteins may vary with cell type and stimulus. The results here demonstrate that PIN1 depletion with shRNA increased iNOS by a posttranscriptional mechanism in LPS-IFN{gamma}-treated MAEC.

Consistent with the increased level of iNOS, knockdown of PIN1 increased the accumulation of nitrite in the medium (Fig. 4A). The increases in NO production and/or iNOS were functionally significant since LPS-IFN{gamma} caused cell injury in KD MAEC, which was antagonized by a NO synthase inhibitor L-NAME (Fig. 4B). The loss of cell adherence is a delayed response, since none was seen at 24 h and was even greater after 72 h (not shown). Interestingly, DETA NONOate, an NO donor, induced detachment only at high millimolar concentrations, and KD and control shRNA cells were equally susceptible (not shown). This suggests that additional factors are required for detachment induced by LPS-IFN{gamma}. For example, NO from iNOS may combine with superoxide from iNOS or other sources, or with other molecules, generating increased levels of agents more toxic than NO in KD cells (16, 31, 32, 46). PIN1 suppression may also sensitize MAEC to the detaching effects of LPS-IFN{gamma}. Whereas the exact mediators of cell detachment remain to be determined, the results indicate that PIN1 suppression increased the induction of iNOS and NO synthase-dependent effects in MAEC.

Calpain and proteasomes degrade iNOS in various cell types (30, 42). Here, depletion of PIN1 stabilized iNOS protein in CHX-treated MAEC (Fig. 5). Furthermore, the calpain inhibitor zVF-CHO enhanced the induction of iNOS by LPS-IFN{gamma} and antagonized the loss of iNOS after inhibition of protein synthesis (Fig. 6). In contrast, the selective proteasome inhibitor epoxomicin (26) did not raise iNOS levels or slow its loss after CHX. Epoxomicin at 2 µM was effective against proteasome activity, since acute loss of I{kappa}B{alpha} and β caused by LPS-IFN{gamma} and induction of iNOS were partially blocked when it was applied before LPS-IFN{gamma} (Fig. 7). The degree of prevention of the loss of I{kappa}B, however, was apparently sufficient to prevent iNOS induction in epoxomicin-treated cells. The overall results indicate that depletion of PIN1 inhibited iNOS degradation and that degradation was mainly due to calpain, as opposed to proteasomal activity, in MAEC treated with LPS-IFN{gamma}.

iNOS was ubiquitinated in A549 and bronchial epithelial cells treated with cytokines, in RAW 264.7 cells treated with LPS-IFN{gamma}, and in HEK293 cells transfected with the human enzyme (19, 30). Degradation of iNOS was antagonized by proteasome inhibitors in HEK293 cells transfected with iNOS, and in RAW 264.7 cells and RT4 epithelial cells treated with LPS or a cytokine mixture for 48 h (19, 30). On the other hand, a glucocorticoid agonist reduced iNOS levels in IFN{gamma}-treated RAW 264.7 cells via calpain activation (42). Interestingly, exogenous treatment of HEK293 cells with the endogenous calpain inhibitor protein calpastatin (6) did not affect degradation of transfected iNOS (30). This suggests that proteasomes, but not calpain, contributed to turnover of transfected iNOS in HEK293 cells. Thus cell type, stimulus, level, or duration of induction, and other factors may affect the iNOS turnover path that operates in different systems.

In vitro proteolysis of iNOS by calpain I is modulated by protein interactions and conformation. Monomers of iNOS were more sensitive to calpain compared with dimers. Degradation was also antagonized by calmodulin, which associates with murine iNOS at a well-characterized site between amino acids 503 and 532 (39, 43). Conformation outside this domain is another factor since heating of an iNOS mutant, lacking the calmodulin-binding sequence, reduced its susceptibility to calpain I, whereas degradation of wild-type iNOS was insensitive to heat treatment (43). PIN1 may regulate iNOS conformation or protein interactions that affect degradation by calpain.

PIN1 might affect iNOS turnover by association with the enzyme, which is phosphorylated (8), and contains several S/T-P motifs that could be WW ligands. iNOS was coimmunoprecipitated with PIN1 using an anti-PIN1 antibody (Fig. 8A). In vitro pulldowns with GST-PIN1 and domain mutants indicated that the WW domain was critical, whereas the K64A catalytic mutant was able to precipitate detectable iNOS (Fig. 8B). Interaction between PIN1 and iNOS could be required for effects of PIN1 on iNOS turnover. However, PIN1 could mediate, or regulate, the association of iNOS with itself or other cofactors, like calmodulin or heat shock protein 90 (43, 51). PIN1 could also stimulate calpain or reduce inhibition by endogenous calpastatin (6). These possibilities are under investigation.

PIN1 is known to affect the expression and function of its various substrates by many mechanisms (44). The range of mechanisms include regulation of transcription, mRNA turnover, enzyme activity, sensitivity to phosphatases, and unbiquitination (4, 5, 28, 34, 35, 38, 49). The findings presented here suggest that PIN1 significantly affects iNOS protein turnover in MAEC that apparently rely on calpain for its degradation. Thus PIN1 may play a significant role in NO synthase-mediated disease or drug actions depending on cell type and stimulus.

In conclusion, suppression of PIN1 increased the induction of iNOS without affecting the mRNA. Instead, PIN1 knockdown antagonized the degradation of iNOS in MAEC, which was sensitive to a calpain inhibitor but not a proteasome inhibitor. WW interactions with p(S/T)-P motifs and proline isomerization may control the sensitivity of iNOS to calpain or the function of the calpain/calpastatin system in MAEC.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. G. Hoyt, Div. of Pharmacology, The Ohio State Univ. College of Pharmacy, 500 West Twelfth Ave., Columbus, OH 43210. http://www.ajpcell.org

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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