Arkadiusz Surażyński

Prolidase-dependent regulation of collagen biosynthesis.

Prolidase [EC.3.4.13.9] is a cytosolic imidodipeptidase, which specifically splits imidodipeptides with C-terminal proline or hydroxyproline. The enzyme plays an important role in the recycling of proline from imidodipeptides (mostly derived from degradation products of collagen) for resynthesis of collagen and other proline-containing proteins. The enzyme activity is up-regulated by β1-integrin receptor stimulation. The increase in the enzyme activity is due to its phosphorylation on serine/threonine residues. Collagen is not only structural component of extracellular matrix. It has been recognized as a ligand for integrin receptors, which play an important role in signaling that regulate ion transport, lipid metabolism, kinase activation and gene expression. Therefore, changes in the quantity, structure and distribution of collagens in tissues may affect cell signaling, metabolism and function. Several line of evidence suggests that prolidase activity may be a step-limiting factor in the regulation of collagen biosynthesis. It has been shown in different physiologic and pathologic conditions. It is of great importance during wound healing, inflammation, aging, tissue fibrosis and possibly skeletal abnormalities seen in Osteogenesis Imperfecta. The mechanism of prolidase-dependent regulation of collagen biosynthesis was found at both transcriptional and post-transcriptional levels. In this study, we provide evidence for prolidase-dependent transcriptional regulation of collagen biosynthesis. The mechanism was found at the level of NF-kB, known inhibitor of type I collagen gene expression. Modulation of integrin-dependent signaling by stimulatory (i.e. thrombin) or inhibitory (i.e. echistatin) β1-integrin ligands or by nitric oxide donors (i.e. DETA/NO) affect prolidase at post-transcriptional level. All those factors may represent novel approach to pharmacotherapy of connective tissue disorders.

Serum and tissue level of insulin‐like growth factor‐I (IGF‐I) and IGF‐I binding proteins as an index of pancreatitis and pancreatic cancer

Previously we have found deregulation of collagen metabolism in human pancreatitis and pancreatic cancer tissues. Insulin‐like growth factor‐I (IGF‐I) is known to stimulate collagen biosynthesis through interaction with IGF‐I receptor. IGF‐I binding proteins (BPs) regulate the activity of IGF‐I. We investigated whether serum and tissue IGF‐I and IGF‐BPs as well as tissue IGF‐I receptor expression may reflect disturbances of collagen metabolism in patients with pancreatitis and pancreatic cancer. In pancreatitis tissue, a significant increase in IGF‐I and IGFBP‐3 content was accompanied by a distinct increase in IGF‐I receptor expression, compared to control pancreas tissue. In contrast, serum from patients with pancreatitis did not show significant increases in IGF‐I and IGFBP‐3 levels, however, significant increases in IGFBP‐1 level (2.5 fold). Moreover, a distinct decrease in radioactive IGF‐binding to the BPs, compared to control serum, was found. Pancreatic cancer tissue and serum of patients with pancreatic cancer showed significant increases in IGF‐I, IGFBP‐3 and IGFBP‐1 content, accompanied by dramatic increases in IGF‐I tissue receptor expression, compared to controls. In serum of patients with pancreatic cancer distinct increases in radioactive IGF‐binding to 46 kDa BP, compared to control serum, were observed. The data suggest that disturbances in tissue collagen metabolism during pancreatic diseases may result from deregulation of IGF‐I homeostasis and that elevated serum levels of IGF‐I, IGFBP‐3 and IGFBP‐1 may serve as markers of pancreatic cancer.

Extracellular matrix and HIF‐1 signaling: The role of prolidase

Hypoxia‐inducible factor‐1 (HIF‐1) plays an important role in stress‐responsive gene expression. Although primarily sensitive to hypoxia, HIF‐1 signaling can be regulated by a number of stress factors including metabolic stress, growth factors and molecules present in the extracellular matrix (ECM). Degradation of ECM by metalloproteinases (MMP) is important for tumor progression, invasion and metastasis. ECM is predominantly collagen, and the imino acids (Pro and HyPro) comprise 25% of collagen residues. The final step in collagen degradation is catalyzed by prolidase, the obligate peptidase for imidodipeptides with Pro and HyPro in the carboxyl terminus. Defective wound healing in patients with inherited prolidase deficiency is associated with histologic features of angiopathy suggesting that prolidase may play a role in angiogenesis. Because HIF‐1α is central to angiogenesis, we considered that prolidase may modulate this pathway. To test this hypothesis, we made expression constructs of human prolidase and obtained stable transfectants in colorectal cancer cells (RKO). Overexpression of prolidase resulted in increased nuclear hypoxia inducible factor (HIF‐1α) levels and elevated expression of HIF‐1−dependent gene products, vascular endothelial growth factor (VEGF) and glucose transporter‐1 (Glut‐1). The activation of HIF‐1‐dependent transcription was shown by prolidase‐dependent activation of hypoxia response element (HRE)‐luciferase expression. We used an oxygen‐dependent degradation domain (ODD)‐luciferase reporter construct as a surrogate for HIF‐1α as an in situ prolyl‐hydroxylase assay. Since this reporter is degraded by VHL‐dependent mechanisms, the increased levels of luciferase observed with prolidase expression reflected the decreased HIF‐1α prolyl hydroxylase activity. Additionally, the differential expression of prolidase in 2 breast cancer cell lines showed prolidase‐dependent differences in HIF‐1α levels. These findings show that metabolism of imidodipeptides by prolidase plays a previously unrecognized role in angiogenic signaling.

Collagen metabolism disturbances are accompanied by an increase in prolidase activity in lung carcinoma planoepitheliale

One of the consequences of neoplastic transformation is deregulation of tissue collagen metabolism. Although metalloproteinases initiate the breakdown of collagen in lung carcinoma, the final step of collagen degradation is mediated by prolidase (E.C.3.4.13.9). We investigated whether prolidase activity could reflect disturbances of collagen metabolism in human lung carcinoma planoepitheliale (Ca pl.). Ten human lung Ca pl. and 10 samples of normal lung parenchyma were compared with respect to prolidase activity and expression (western immunoblot), the content of collagen and collagen degradation products (free and bound hydroxyproline determination), β1 integrin subunit expression (western immunoblot) and collagenolytic activity (zymography). An increase in collagen content (66%, P < 0.05), free proline pool (50%, P < 0.05) and collagenolytic activity was accompanied by a significant increase in the prolidase activity (106%, P < 0.05) and its expression in Ca pl. No differences were found between Ca pl. and the control lung tissue with respect to β1 integrin expression. Prolidase activity may reflect disturbances in tissue collagen metabolism in lung Ca pl. and it may, therefore, serve as a sensitive marker of the disease.

Nitric oxide regulates prolidase activity by serine/threonine phosphorylation.

Prolidase [E.C. 3.4.13.9], a member of the matrix metalloproteinase (MMP) family, is a manganese‐dependent cytosolic exopeptidase that cleaves imidodipeptides containing C‐terminal proline or hydroxyproline. It plays an important role in collagen metabolism, matrix remodeling and cell growth. Nitric oxide (NO), a versatile signaling molecule, regulates many processes including collagen synthesis and matrix remodeling and, thereby, may modulate angiogenesis, tumor invasiveness, and metastasis. Thus, we considered that prolidase may be an important target of NO regulation. In our study, SIN I and DETA/NO were used as NO donors. Both donors increased prolidase activity in a time‐dependent and dose‐dependent manner. Prolidase activity increased not only with NO donors but also with endogenous NO in cells transfected with iNOS. The effect of iNOS was abolished by treatment with S‐methylisothiourea (SMT), a selective inhibitor of iNOS. However, with either exogenous or endogenous sources of NO, the increase in prolidase activity was not accompanied by increased prolidase expression. Therefore, we suspected phosphorylation of prolidase as a potential mechanism regulating enzyme activation. We observed increased serine/threonine phosphorylation on prolidase protein in cells treated with NO donors and in cells transfected with iNOS. To determinate the pathways that may mediate prolidase induction by NO, we first used 8‐Br‐cGMP, a cGMP agonist, and found that 8‐Br‐cGMP strongly and rapidly stimulated prolidase activity accompanied by increased phosphorylation. Rp‐8‐Br‐pCPT‐cGMP, an inhibitor of cGMP, reduced NO donor‐stimulated prolidase activity to control levels. To test wheher the MAPK pathway is involved in this NO‐dependent activation, we used an ERK1/2 inhibitor and found that it had no effect on prolidase activity increased by NO donors. These results demonstrate that NO stimulates prolidase activity by increasing serine/threonine phosphorylation through PKG‐cGMP pathway, but independent of MAPK and suggest an interaction between inflammatory signaling pathways and regulation of the terminal step of matrix degradation. J. Cell. Biochem.

Estrogenic and antiestrogenic effects of raloxifene on collagen metabolism in breast cancer MCF-7 cells

We compared the effects of different concentrations of raloxifene (1 ,4 and 10 μM) on collagen biosynthesis ,gelatinolytic and prolidase activities and matrix metalloproteinase (MMP) expression (MMP-2 and MMP-9) in estradiol-stimulated (2 nM) breast cancer MCF-7 cells. Raloxifene inhibited in a dose-dependent manner the proliferation of MCF-7 cells ,independently of the presence or absence of estradiol in the growth medium. Raloxifene at concentrations of 1 μM and 4 μM inhibited collagen biosynthesis by about 10-fold and prolidase activity by about 50% ,while at a concentration of 10 μM it inhibited these processes by only about 25%. This phenomenon was accompanied by differences in gelatinolytic activity and MMP (MMP-2 and MMP-9) expression as demonstrated by zymography and Western immunoblot analysis, respectively. In estrogenstimulated MCF-7 cells ,cultured in the presence of 1 μM raloxifene ,a dramatic increase in the activity of both collagenases was found. In contrast ,addition of raloxifene at a concentration of 10 μM to the medium of the cells resulted in restoration of gelatinolytic activity to that found in control cells. Similarly ,but at both doses (1 and 10 μM) ,raloxifene was able to reduce MMP-2 expression in the cells. However ,when used alone (without estradiol) a concentration of 1 μM raloxifene strongly stimulated MMP-2 expression ,while at a concentration of 10 μM the effect was not observed. In the case of MMP-9 ,only trace amounts of this gelatinase were detected ,although in contrast to MMP-2 ,an increase in its expression was noticed at a concentration of 10 μM raloxifene. The data raise the possibility that in estrogen-stimulated MCF-7 cells ,raloxifene at low concentrations (1 and 4 μM) evokes antiestrogenic effect on collagen biosynthesis and prolidase activity on the one hand ,and an estrogenic effect on gelatinolytic activity on the other ,while at higher concentrations (about 10 μM) it evokes an estrogenic effect on collagen biosynthesis and prolidase activity, and an antiestrogenic effect on gelatinolytic activity. Our data suggest that the effects of raloxifene on collagen synthesis ,prolidase and metalloproteinase activities in breast cancer may explain its role in the prevention of breast cancer development.

Melanin potentiates daunorubicin-induced inhibition of collagen biosynthesis in human skin fibroblasts

One of the recognized side effects of antineoplastic anthracyclines is poor wound healing, resulting from an impairment of collagen biosynthesis. The most affected tissue is skin. The mechanism underlying the tissue specificity of the side effects of anthracyclines has not been established. In view of the fact that a number of pharmacologic agents are known to form complexes with melanin and melanins are abundant constituents of the skin, we determined whether daunorubicin interacts with melanin and how this process affects collagen biosynthesis in cultured human skin fibroblasts. Results indicated that daunorubicin forms complexes with melanin. Scatchard analysis showed that the binding of daunorubicin to melanin was heterogeneous, suggesting the presence of two classes of independent binding sites with K1 = 1.83 x 10(5) M(-1) and K2 = 5.52 x 10(3) M(-1). The number of strong binding sites was calculated as n1 = 0.158 micromol/mg of melanin and the number of weak binding sites as n2 = 0.255 micromol/mg of melanin. We have suggested that prolidase, an enzyme involved in collagen metabolism, may be one of the targets for anthracycline-induced inhibition of collagen synthesis. We found that daunorubicin induced inhibition of prolidase activity (IC50 = 10 microM), collagen biosynthesis (IC50 = 70 microM) and DNA biosynthesis (IC50= 10 microM) in human skin fibroblasts. Melanin (100 microg/ml) by itself produced about 25% inhibition of DNA synthesis and prolidase activity but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 microg/ml) to daunorubicin-treated cells (at IC50 concentration) augmented the inhibitory action of daunorubicin on collagen and DNA biosynthesis without having any effect on prolidase activity. The same effect was achieved when the cells were treated with daunorubicin at one-fourth of the IC50 given at 0, 6, 12 and 18 h during a 24-h incubation. The data suggest that the melanin-induced augmentation of the inhibitory effects of daunorubicin on collagen and DNA biosynthesis may result from: (i) accumulation of the drug in the extracellular matrix, (ii) gradual dissociation of the complex, and (iii) constant action of the released drug on cell metabolism. The phenomenon may explain the potential mechanism for the organ specificity of daunorubicin-induced poor wound healing in patients administered this drug.

Phosphorylation of prolidase increases the enzyme activity

Prolidase [EC 3.4.13.9] is a ubiquitously distributed imidodipeptidase that catalyzes the hydrolysis of C-terminal proline-containing dipeptides. The enzyme plays an important role in the recycling of proline for collagen synthesis and cell growth. Although, the increase in the enzyme activity is correlated with increased rate of collagen turnover, the mechanism by which prolidase is regulated remain largely unknown. In the present study we found that phosphorylation of fibroblasts prolidase may be an underlying mechanism for up regulation of the enzyme activity. Supporting evidence comes from the following observations: (1) immunoprecipitated prolidase was detected as a phosphotyrosine protein as shown by western immunoblot analysis, (2) tyrosine kinase inhibitor – erbstatin induced (in a dose dependent manner) a decrease in prolidase activity in cultured human skin fibroblasts, (3) anti-phosphotyrosine antibody reduced and phosphotyrosine phosphatase 1B antibody (anti-PTP 1B) increased (in a dose dependent manner) the prolidase activity in extract of fibroblasts homogenate, (4) decrease in prolidase activity from collagenase treated or serum starved fibroblasts can be partially prevented by incubating fibroblasts homogenate extract with anti-PTP 1B antibody. These results provide evidence that prolidase is phosphotyrosine enzyme and suggest that the activity of prolidase may be up regulated by the enzyme phosphorylation.

Protective effect of hyaluronic acid on interleukin-1-induced deregulation of β1-integrin and insulin-like growth factor-I receptor signaling and collagen biosynthesis in cultured human chondrocytes

The mechanism of protective action of hyaluronic acid (HA) on collagen metabolism disturbances in tissues during inflammation is not known. Insulin-like growth factor-I (IGF-I) receptor and β1-integrin receptor signaling plays an important role in the regulation of collagen biosynthesis at both transcriptional and post-transcriptional levels. The present study was undertaken to evaluate the effect of IL-1β (inductor of experimental inflammation) on the signaling pathways as well as on collagen biosynthesis, gelatinases and prolidase activity in cultured human chondrocytes and the effect of HA on these processes. It was found that IL-1β-dependent inhibition of collagen biosynthesis was accompanied by increase in β1-integrin receptor, NF-kB expressions, and increase in phosphorylation of FAK, that resulted in stimulation of metalloproteinase MMP-2 and MMP-9 activities, but not prolidase activity and expression. Simultaneously, decrease in expression of IGF-I receptor and phosphorylation of Akt and p38 were found. All those processes were counteracted by HA. This suggests that cross talk between β1-integrin and IGF-I receptors is disturbed by IL-1β, and HA recovers their proper signaling in cultured chondrocytes. We propose that IGF-I receptor and β1-integrin signaling may play an important role in protective effect of hyaluronic acid on interleukin-1-induced inhibition of collagen biosynthesis in cultured human chondrocytes.

Cytotoxic efficacy of a novel dinuclear platinum(II) complex in human breast cancer cells.

Evaluation of the cytotoxicity of a novel dinuclear platinum(II) complex of formula Pt(2)(2-picoline)(4)(berenil)(2) employing a MTT assay and inhibition of [(3)H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that the complex was more of a potent antiproliferative agent than cisplatin. The DNA-binding ability of Pt(2)(2-picoline)(4)(berenil)(2) estimated by an ethidium displacement assay indicated that the complex showed strong specificity for AT base pairs in the minor groove of DNA. Our study showed that Pt(2)(2-picoline)(4)(berenil)(2) was a potent catalytic inhibitor of topoisomerase II in opposition to cisplatin. Pt(2)(2-picoline)(4)(berenil)(2) was found to be a more active inhibitor of collagen biosynthesis than cisplatin. The up regulation of beta(1)-integrin and insulin-like growth factor I (IGF-I) receptor expression by the complex was shown to be accompanied by an increase in the expression of mitogen activated protein kinases in breast cell lines. The phenomenon was related to the increased expression of nuclear factor-kappaB (NuF-kappaBeta) by Pt(2)(2-picoline)(4)(berenil)(2) as shown by the Western immunoblot analysis. Flow cytometric analysis and a fluorescent microscopy assay demonstrated that cell death appeared to result from apoptosis, with the possibility of secondary necrosis. The data presented suggested that Pt(2)(2-picoline)(4)(berenil)(2) impaired growth and metabolism of breast cancer cells more efficiently than cisplatin. These results indicated also the different properties of Pt(2)(2-picoline)(4)(berenil)(2) and cisplatin.