Expression of plasminogen activator inhibitors type 1 and type 3 and urokinase plasminogen activator protein and mRNA in breast cancer.

BACKGROUND: The urokinase plasminogen activator (uPA) system has been involved in cancer cell invasion and in metastasis. uPA activity is controlled by its principal inhibitor, the PA inhibitor type-1 (PAI-1), but it can also be inhibited by PAI-3. Increased levels of uPA and PAI-1 are known to be associated with a poor prognosis in breast cancer. To our knowledge this is the first study of the expression and role of PAI-3 in human breast cancer tissue. MATERIALS AND METHODS: Protein and mRNA levels were evaluated for uPA, PAI-1 and PAI-3 in breast cancer tissues from 70 different patients. The localization of antigen and mRNA of these proteins was studied by immunohistochemistry and in situ hybridization, respectively. RESULTS: No significant differences were observed for PAI-3 mRNA or protein levels between the nodal status groups or the different post-surgical tumor-node-metastasis (pTNM) stages. However, uPA and PAI-1 mRNA and antigen levels significantly increased at the pTNM stage and in node-positive patients. PAI-3 antigen levels were significantly higher in early relapse-free patients, whereas PAI-1 antigen levels were significantly higher in patients who suffered a relapse. PAI-3 protein and mRNA were localized in stromal cells. PAI-1 and uPA protein were detected in cancer, endothelial and stromal cells and their mRNA mainly in stromal cells. CONCLUSIONS: Our results indicate that PAI-3 is expressed in human breast cancer tissues, and that elevated levels of PAI-3 could be a positive prognostic factor in this disease. A potential mechanism for the contribution of PAI-3 to a positive long-term outcome may involve suppression of tumor invasion through protease inhibition in stroma.

Immunolocalization of protein C inhibitor in differentiation of human epidermal keratinocytes.

Keratinocytes propagated in low calcium (0.05 mM) serum-free medium grow as monolayers and exhibit morphological and biosynthetic phenotypes similar to the keratinocytes of the basal layer in normal epidermis. When the calcium in the medium is increased to 1.5 mM, the keratinocytes start to stratify and differentiate. Such differentiation is important in the formation of an epidermal barrier. Proteolysis plays a crucial role in the process. The functions of most of the plasminogen activator cascade components in human skin have been studied, but little was known about the expression and role of protein C inhibitor in the differentiation of human epidermal keratinocytes. In the present study, we used immunohistochemistry and immunocytochemistry to examine the immunolocalization of protein C inhibitor in normal human skin and in cultured keratinocytes in serum-free medium with low and high calcium, respectively. The results indicated that protein C inhibitor is mainly localized in superficial and more differentiated keratinocytes in normal human epidermis. Keratinocytes positive for protein C inhibitor were detected in cultures containing both low and high calcium media, and the level of protein C inhibitor was increased in high calcium medium. This increase was accompanied by an altered intracellular distribution, from the perinuclear cytoplasm in undifferentiated keratinocytes to the whole cytoplasm in differentiated keratinocytes. Further study revealed that protein C inhibitor was incorporated into the cornified envelope in normal skin keratinocytes and cultured differentiated keratinocytes. Our results suggest that protein C inhibitor may be involved in the differentiation of keratinocytes.

Protein C inhibitor expression by adult rat Sertoli cells: effects of testosterone withdrawal and replacement.

Protein C inhibitor (PCI), a member of the plasma serine protease inhibitor family, has been reported to be abundantly expressed in the seminal vesicles and testes. In this study, we examine the localization and regulation of the PCI gene and protein expression in testes and freshly isolated Sertoli cells from control rats, rats treated with luteinizing hormone-suppressive testosterone/estradiol (TE)-containing Silastic capsules for 7, 14, 28, and 56 days, and rats treated with TE for 56 days, followed by high levels of testosterone for 7 or 14 days. The administration of the TE capsules for 56 days resulted in reduced testicular testosterone, from approximately 100 ng/mL in the controls to approximately 10 ng/mL, accompanied by a 73% reduction in testicular weight. PCI mRNA levels in freshly isolated Sertoli cells were reduced by 30% and 54% following TE treatment for 28 and 56 days, respectively. When rats that had received TE capsules for 56 days were provided replacement testosterone, there was a 40% increase in PCI mRNA levels within 7 days in the absence of any change in testicular weight, and PCI mRNA levels returned to control values by 14 days. The decrease in PCI mRNA levels in TE-treated rats was paralleled by a decrease in PCI protein levels in whole testis lysates and in seminiferous tubule fluid (STF). Protease activity was significantly increased in STF following 56 days of TE treatment. Taken together, these results indicate that 1) PCI in the testis is expressed by Sertoli cells; 2) the testicular expression of PCI is responsive to intratesticular testosterone levels; and 3) protease activity within the seminiferous epithelium is elevated when intratesticular concentration is decreased, perhaps as a consequence of decreased PCI.

Molecular cloning, tissue distribution and androgen regulation of rat protein C inhibitor.

Protein C inhibitor (PCI) is the plasma serine protease inhibitor of activated protein C, the active enzyme of the anticoagulant protein C pathway. Recently, PCI was also detected in human seminal plasma and reproductive organs (testis, seminal vesicle and prostate) suggesting that PCI may also play an important role in the reproductive system. In this study, we cloned the full length of rat PCI cDNA, and determined its amino acid sequence and tissue distribution. We also evaluated the effect of androgen on PCI mRNA expression in seminal vesicles and testes. The isolated 2074-bp rat PCI cDNA was composed of a 47-bp 5'-non-coding region, a 1218-bp coding region of a 406-amino acid precursor protein, a stop codon and a 806-bp 3'-non-coding region. The deduced amino acid sequence of rat PCI showed 85.7%, 64.1% and 62.2% homology with that of mouse, rhesus monkey and human PCIs, respectively. Northern blot analysis showed that the rat PCI mRNA is expressed strongly in the seminal vesicle, moderately in the testis, but not in the liver. PCI mRNA expression in seminal vesicles and testes was found to increase during the process of development, suggesting that it is under androgen control. Subsequently, we examined the effect of castration and/or treatment with 17beta-estradiol or testosterone on PCI mRNA expression in the mature rat seminal vesicles. The PCI mRNA expression in seminal vesicles was significantly decreased after castration or 17beta-estradiol treatment. Testosterone itself did not affect PCI mRNA expression, but treatment in castrated rats significantly enhanced its mRNA expression. These findings suggest that the PCI gene expression in rat seminal vesicles is regulated by androgen.

Regulation of protein C inhibitor (PCI) activity by specific oxidized and negatively charged phospholipids.

Protein C inhibitor (PCI) is a serpin with affinity for heparin and phosphatidylethanolamine (PE). We analyzed the interaction of PCI with different phospholipids and their oxidized forms. PCI bound to oxidized PE (OxPE), and oxidized and unoxidized phosphatidylserine (PS) immobilized on microtiter plates and in aqueous suspension. Binding to OxPE and PS was competed by heparin, but not by the aminophospholipid-binding protein annexin V or the PCI-binding lipid retinoic acid. PS and OxPE stimulated the inhibition of activated protein C (aPC) by PCI in a Ca(++)-dependent manner, indicating that binding of both, aPC (Ca(++) dependent) and PCI (Ca(++) independent), to phospholipids is necessary. A peptide corresponding to the heparin-binding site of PCI abolished the stimulatory effect of PS on aPC inhibition. No stimulatory effect of phospholipids on aPC inhibition was seen with a PCI mutant lacking the heparin-binding site. A heparin-like effect of phospholipids (OxPE) was not seen with antithrombin III, another heparin-binding serpin, suggesting that it is specific for PCI. PCI and annexin V were found to be endogenously colocalized in atherosclerotic plaques, supporting the hypothesis that exposure of oxidized PE and/or PS may be important for the local regulation of PCI activity in vivo.

Protein C inhibitor (plasminogen activator inhibitor-3) expression in the CWR22 prostate cancer xenograft.

The serine protease inhibitor (serpin) protein C inhibitor (PCI) has been found in the prostate and possibly is a marker to distinguish normal prostate, benign prostatic hyperplasia, and prostate cancer. In this study, we assessed PCI expression in normal, hyperplastic, and malignant prostatic tissues, prostate cancer cell lines, and the CWR22 prostate cancer xenograft model that allowed us to study PCI expression and its regulation in response to androgens. By Northern blot, immunohistochemistry, and in situ hybridization, we found that PCI was expressed in both benign and malignant prostate tissues. Protein C inhibitor was expressed in both androgen-independent (PC-3) and androgen-dependent (LNCaP) prostate cancer cell lines. Furthermore, PCI was detected in all CWR22 tumor samples (androgen dependent, 6 days post-castration, 12 days post-castration followed by 72 h of testosterone treatment, and recurrent CWR22 tumor), although expression of the mature forms of both prostate-specific antigen (PSA) and its homolog, kallikrein 2 (hK2), was clearly androgen-dependent. These results suggest that PCI expression is not regulated by androgens and that PCI is unlikely to be a tumor suppressor gene, but also that PCI may be involved in regulating key serine proteases involved in metastatic prostate disease.

Synthesis and ultrastructural localization of protein C inhibitor in human platelets and megakaryocytes.

The occurrence of protein C inhibitor (PCI) in human platelets and megakaryocytes was analyzed. As judged from enzyme-linked immunosorbent assays (ELISAs), PCI was present in platelets at a concentration of 160 ng/2 x 10(9) cells. Its specific activity was 5 times higher than that of plasma PCI. Consistently, mainly the 57-kD form (active PCI) and some high molecular weight (M(r)) forms, but no bands corresponding to cleaved PCI, were detected when platelet lysates were immunoprecipitated with monoclonal anti-PCI-IgG and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The localization of PCI in platelets was studied by immunofluorescence histochemistry and immunotransmission electron microscopy: PCI was detected in alpha granules, in the open canalicular system, and on the plasma membrane. At these sites, colocalization with plasminogen activator inhibitor-1 was seen. Studies were performed to clarify whether platelet PCI is endogenously synthesized or taken up from plasma. Internalization of biotinylated-PCI was analyzed using platelets in suspension and gold-labeled streptavidin for visualization of incorporated biotin. Dose- and time-dependent uptake of PCI was found. PCI mRNA was detected in platelets by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting, as well as in megakaryocytes by in situ hybridization of human bone marrow cryosections. We therefore conclude that platelets contain a functionally active PCI pool that is derived from both endogenous synthesis as well as internalization.

Mutagenesis of recombinant protein C inhibitor reactive site residues alters target proteinase specificity.

Protein C inhibitor (PCI) is a heparin-binding plasma serine proteinase inhibitor (serpin) which is thought to be a physiological regulator of activated protein C. We are using recombinant PCI (rPCI) to study structural determinants of target proteinase specificity. A cDNA encoding full-length PCI has been expressed as a fully active proteinase inhibitor using Autographa californica nuclear polyhedrosis virus (baculovirus). rPCI was expressed maximally 4 days after infection and could be expressed either in Sf9 or High-Five cells. rPCI bound heparin and was conveniently purified with heparin-Sepharose (eluting > 0.5 M NaCl). The rPCI formed sodium dodecyl sulfate-polyacrylamide gel electrophoresis-stable complexes with thrombin and activated protein C (APC). The inhibitory properties of wild-type rPCI and plasma-derived PCI are essentially the same either in the absence or presence of heparin with thrombin, APC, trypsin, and urokinase. The residues Phe353-Arg354-Ser355 (P2-P1-P1') constitute part of the reactive site loop of PCI with the Arg-Ser peptide bond being cleaved by the proteinase. Using site-directed mutagenesis we studied the contribution of the reactive site FRS for proteinase inhibition in rPCI. Changing the P1 residue Arg354-->Met generated a reactive site similar to alpha 1-proteinase inhibitor which was a much poorer inhibitor of thrombin, APC, trypsin, and urokinase. Changing the P2 residue Phe353-->Gly generated a mutant with a reactive site like antithrombin which was better at inhibiting thrombin or urokinase, but was much less active with APC or trypsin. Changing the P1' residue Ser355-->Met generated a reactive site like plasminogen activator inhibitor-1 and this protein inhibits all the proteinases essentially like wild-type rPCI. These results show the importance of PCI's Phe353 (P2) and Arg354 (P1) in target proteinase specificity, and they further support the concept of reactive site sequences determining serpin function.

Activated protein C-protein C inhibitor complex formation: characterization of a neoepitope provides evidence for extensive insertion of the reactive center loop.

Protein C inhibitor, a serine proteinase inhibitor (serpin), is the physiologically most important inhibitor of activated protein C. We have made a monoclonal antibody (M36) that binds with equally high affinity to an epitope present in activated protein C-protein C inhibitor complexes and cleaved loop-inserted protein C inhibitor. Insertion of a synthetic N-acetylated tetradecapeptide (corresponding to residues P1-P14 of the reactive center loop) into beta-sheet A of the uncleaved inhibitor also exposed the epitope. The antibody had no apparent affinity for native uncleaved inhibitor or for the free peptide. Synthetic P1-P14 analogues, with Arg P13 or Ala P9 substituted to the residues found in mouse protein C inhibitor (Thr and Ile, respectively), were also inserted in beta-sheet A. The Arg P13/Thr substitution led to a greatly impaired reactivity with the antibody, whereas the Ala P9/Ile mutation resulted in a modest loss of reactivity with the antibody. These results indicate that complex formation leads to insertion of the reactive center loop in beta-sheet A from Arg P14 and presumably beyond Ala P9. Moreover, to the best of our knowledge, this is the first instance where the neoepitope of a complexation-specific monoclonal antibody has been localized to the loop-inserted part of beta-sheet A, the part of the serpin where the complexation-induced conformational change is most conspicuous.

Expression of protein C inhibitor (PCI) in benign and malignant prostatic tissues.

BACKGROUND: Protein C inhibitor (PCI) occurs at high concentration in seminal plasma, and inhibits human glandular kallikrein-2 and, less readily, prostate-specific antigen. Previous studies have localized PCI in the male genital tract. Here we have performed a detailed investigation of PCI expression in the prostatic tissues, metastases, and cell lines. METHODS: Immunohistochemistry, in situ hybridization, and Western blotting were used to study prostatic tissues, metastases, and PC-3, DU-145, and LNCaP cells. RESULTS: PCI was immunolocalized in tissue microarray spots with BPH epithelium (detection rate 100%), PIN lesions (100%), tumors (96%), metastases (88%), and in all cell lines. ISH and WB supported the findings. CONCLUSIONS: PCI is widely expressed in benign prostatic epithelium, and may act as a local regulator of enzymatic activity in seminal fluid, of importance for normal sperm function. Lack of PCI expression in a subpopulation of high-grade tumor cells in combination with maintained protease expression may facilitate invasive growth patterns.

cis-elements required for expression of human protein C inhibitor gene in HepG2 cells and its androgen-dependent expression in rat reproductive organs.

Protein C inhibitor (PCI) is a plasma serine protease inhibitor of activated protein C, which is the main protease of the anticoagulant protein C pathway. Human PCI is synthesized in the liver, kidney, and several reproductive organs (testis, seminal vesicle, and prostate). In the present study, we characterized cis-elements of the human PCI gene required for expression in the hepatoma-derived cell line, HepG2 cells, and also evaluated rat PCI mRNA expression, particularly on the effect of androgen in rat reproductive tissues. On the PCI gene expression in HepG2 cells, transient expression assays using several deletion mutants and site-directed mutants of the human PCI gene and gel mobility shift assays using several synthetic oligonucleotides showed that the Spl-binding site (residues -302 to -294) and the upstream AP2-binding site (residues -350 to -343) play roles as the promoter and the enhancer, respectively. Both the A-activator-binding site (residues -422 to -414) and the interferon-gamma response element (residues -164 to -157) serve as the silencer. In the study on PCI mRNA expression in the reproductive organs, we first cloned rat PCI cDNA and then evaluated the effect of androgen on the PCI mRNA expression. The isolated rat PCI cDNA contained a 1,218-bp coding region of a 406-amino acid precursor protein. The deduced amino acid sequence of rat PCI showed an 85.7% and 62.2% homology with that of mouse and human PCIs, respectively. Northern blot analysis showed that rat PCI mRNA was strongly expressed in the seminal vesicles, moderately in the testes, but not in the liver. PCI mRNA expression in seminal vesicles and testes increased during the process of development. The PCI mRNA expression in seminal vesicles was significantly decreased after castration or after 17beta-estradiol treatment. Treatment with testosterone in the castrated rats significantly enhanced its mRNA expression. These findings suggest that the PCI gene expression in rat seminal vesicles is under androgen control.