Inhibition Of Collagen Biosynthesis Research Articles

Amaranthus cruentus L. Seed Oil Counteracts UVA-Radiation-Induced Inhibition of Collagen Biosynthesis and Wound Healing in Human Skin Fibroblasts.

The effect of Amaranthus cruentus L. seed oil (AmO) on collagen biosynthesis and wound healing was studied in cultured human dermal fibroblasts exposed to UVA radiation. It was found that UVA radiation inhibited collagen biosynthesis, prolidase activity, and expression of the β1-integrin receptor, and phosphorylated ERK1/2 and TGF-β, while increasing the expression of p38 kinase. The AmO at 0.05-0.15% counteracted the above effects induced by UVA radiation in fibroblasts. UVA radiation also induced the expression and nuclear translocation of the pro-inflammatory NF-κB factor and enhanced the COX-2 expression. AmO effectively suppressed the expression of these pro-inflammatory factors induced by UVA radiation. Expressions of β1 integrin and IGF-I receptors were decreased in the fibroblasts exposed to UVA radiation, while AmO counteracted the effects. Furthermore, AmO stimulated the fibroblast's migration in a wound healing model, thus facilitating the repair process following exposure of fibroblasts to UVA radiation. These data suggest the potential of AmO to counteract UVA-induced skin damage.

Amnion responses to intrauterine inflammation and effects of inhibition of TNF signaling in preterm Rhesus macaque

Intrauterine infection/inflammation (IUI) is a frequent complication of pregnancy leading to preterm labor and fetal inflammation. How inflammation is modulated at the maternal-fetal interface is unresolved. We compared transcriptomics of amnion (a fetal tissue in contact with amniotic fluid) in a preterm Rhesus macaque model of IUI induced by lipopolysaccharide with human cohorts of chorioamnionitis. Bulk RNA sequencing (RNA-seq) amnion transcriptomic profiles were remarkably similar in both Rhesus and human subjects and revealed that induction of key labor-mediating genes such as IL1 and IL6 was dependent on nuclear factor κB (NF-κB) signaling and reversed by the anti-tumor necrosis factor (TNF) antibody Adalimumab. Inhibition of collagen biosynthesis by IUI was partially restored by Adalimumab. Interestingly, single-cell transcriptomics, flow cytometry, and immunohistology demonstrated that a subset of amnion mesenchymal cells (AMCs) increase CD14 and other myeloid cell markers during IUI both in the human and Rhesus macaque. Our data suggest that CD14+ AMCs represent activated AMCs at the maternal-fetal interface.

Proline Dehydrogenase/Proline Oxidase (PRODH/POX) Is Involved in the Mechanism of Metformin-Induced Apoptosis in C32 Melanoma Cell Line.

The role of proline dehydrogenase/proline oxidase (PRODH/POX) in the mechanism of antineoplastic activity of metformin (MET) was studied in C32 melanoma cells. PRODH/POX is a mitochondrial enzyme-degrading proline that is implicated in the regulation of cancer cell survival/apoptosis. The enzyme is activated by AMP kinase (AMPK). It has been found that MET induced a significant decrease in cell viability and DNA biosynthesis accompanied by an increase in the expressions of AMPK and PRODH/POX in C32 cells. The mechanism for MET-dependent cytotoxicity on C32 cells was found at the level of PRODH/POX-induced ROS generation and activation of Caspase-3 and Caspase-9 expressions in these cells. The effects were not observed in MET-treated PRODH/POX knock-out C32 cells. Of interest is an MET-dependent increase in the concentration of proline, which is a substrate for PRODH/POX. This phenomenon is due to the MET-dependent inhibition of collagen biosynthesis, which is the main proline-utilizing process. It has been found that the underlying mechanism of anticancer activity of MET involves the activation of AMPK, PRODH/POX, increase in the cytoplasmic concentration of proline, inhibition of collagen biosynthesis, and stimulation of PRODH/POX-dependent ROS generation, which initiate the apoptosis of melanoma cells.

Nonsteroidal Anti-Inflammatory Drugs as PPARγ Agonists Can Induce PRODH/POX-Dependent Apoptosis in Breast Cancer Cells: New Alternative Pathway in NSAID-Induced Apoptosis

Nonsteroidal anti-inflammatory drugs (NSAIDs) are considered to be therapeutics in cancer prevention because of their inhibitory effect on cyclooxygenases (COX), which are frequently overexpressed in many types of cancer. However, it was also demonstrated that NSAIDs provoked a proapoptotic effect in COX knocked-out cancer cells. Here, we suggest that this group of drugs may provoke antineoplastic activity through the activation of PPARγ, which induces proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis. PRODH/POX is a mitochondrial enzyme that catalyzes proline degradation, during which ATP or reactive oxygen species (ROS) are generated. We have found that NSAIDs induced PRODH/POX and PPARγ expressions (as demonstrated by Western Blot or immunofluorescence analysis) and cytotoxicity (as demonstrated by MTT, cytometric assay, and DNA biosynthesis assay) in breast cancer MCF7 cells. Simultaneously, the NSAIDs inhibited collagen biosynthesis, supporting proline for PRODH/POX-induced ROS-dependent apoptosis (as demonstrated by an increase in the expression of apoptosis markers). The data suggest that targeting proline metabolism and the PRODH/POX–PPARγ axis can be considered a novel approach for breast cancer treatment.

Proline oxidase silencing inhibits p53-dependent apoptosis in MCF-7 breast cancer cells

Proline oxidase (POX) is mitochondrial proline-degrading enzyme of dual apoptosis/survival function. POX expression and proline availability are considered an underlying mechanism for differential POX functions. The mechanism for POX-dependent regulation of cell death/survival was studied in wild-type (MCF-7WT) and shRNA POX-silenced breast cancer cells (MCF-7iPOX). Proline concentration and proteomic analyses were determined by LC/MS/QTOF and LC/MS/ORBITRA, respectively. Inhibition of collagen biosynthesis (proline utilizing process) by 2-methoxyestradiol (2ME) contributed to induction of apoptosis in MCF-7WT cells, as detected by increase in the expression of active caspase-3, -9 and p53. The process was not shown in MCF-7iPOX. In MCF-7iPOX cells prolidase activity and expression as well as proline concentration were drastically increased, compared to MCF-7WT cells. Down-regulation of p53 in MCF-7iPOX cells was corroborated by proteomic analysis showing decrease in the expression of p53-related proteins. The mechanism for down-regulation of p53 expression in MCF-7iPOX cells was found at the level of p53–PEPD complex formation that was counteracted by hydrogen peroxide treatment. In this study, we found that silencing POX modulate pro-survival phenotype of MCF-7 cells and suggest that the mechanism of this process undergoes through down-regulation of p53-dependent signaling.

Collagen metabolism as a regulator of proline dehydrogenase/proline oxidase-dependent apoptosis/autophagy

Recent studies on the regulatory role of amino acids in cell metabolism have focused on the functional significance of proline degradation. The process is catalysed by proline dehydrogenase/proline oxidase (PRODH/POX), a mitochondrial flavin-dependent enzyme converting proline into ∆1-pyrroline-5-carboxylate (P5C). During this process, electrons are transferred to electron transport chain producing ATP for survival or they directly reduce oxygen, producing reactive oxygen species (ROS) inducing apoptosis/autophagy. However, the mechanism for switching survival/apoptosis mode is unknown. Although PRODH/POX activity and energetic metabolism were suggested as an underlying mechanism for the survival/apoptosis switch, proline availability for this enzyme is also important. Proline availability is regulated by prolidase (proline supporting enzyme), collagen biosynthesis (proline utilizing process) and proline synthesis from glutamine, glutamate, α-ketoglutarate (α-KG) and ornithine. Proline availability is dependent on the rate of glycolysis, TCA and urea cycles, proline metabolism, collagen biosynthesis and its degradation. It is well established that proline synthesis enzymes, P5C synthetase and P5C reductase as well as collagen prolyl hydroxylases are up-regulated in most of cancer types and control rates of collagen biosynthesis. Up-regulation of collagen prolyl hydroxylase and its exhaustion of ascorbate and α-KG may compete with DNA and histone demethylases (that require the same cofactors) to influence metabolic epigenetics. This knowledge led us to hypothesize that up-regulation of prolidase and PRODH/POX with inhibition of collagen biosynthesis may represent potential pharmacotherapeutic approach to induce apoptosis or autophagic death in cancer cells. These aspects of proline metabolism are discussed in the review as an approach to understand complex regulatory mechanisms driving PRODH/POX-dependent apoptosis/survival.

Verapamil and collagenase differentially affect collagen metabolism in experimental model of Peyronie's disease

ObjectivesPeyronie's disease (PD) is accompanied by remodelling of connective tissue into fibrotic plaque. Treatment of the inflammatory and fibrotic phases of the disease is not established. The aim of the study was to evaluate the effect of verapamil (VER) and bacterial collagenase (COLL) on collagen metabolism and cell migration in fibroblasts with experimental wound healing and inflammation as an in vitro model of PD. Materials and methodsIn vitro model of PD was designed using experimental model of inflammation induced by Interleukin-1 (IL-1) in cultured fibroblasts and mechanical damage of the cells. Cell viability, cell proliferation, collagen biosynthesis, prolidase activity and cell migration were studied in both models of the cells treated with VER and COLL. ResultsVER decreased cell viability, DNA and collagen biosynthesis and increased prolidase activity in control fibroblast, while in “wounded” fibroblasts it significantly decreased all the processes. COLL did not affect cell viability and DNA biosynthesis, while inhibited collagen biosynthesis and prolidase activity in both control and “wounded” fibroblasts. In IL-1-treated fibroblasts VER inhibited all studied processes except prolidase activity, while COLL inhibited only collagen biosynthesis and prolidase activity. COLL accelerated cell migration, while VER attenuated the process in fibroblast model of wound healing, compared to control cells. ConclusionVER and COLL attenuate collagen biosynthesis in both fibroblast models. The VER-dependent inhibition of collagen biosynthesis was accompanied by inhibition of DNA biosynthesis at high prolidase activity, while COLL affected this process through inhibition of prolidase activity at high rate of DNA biosynthesis. It shows that anti-fibrotic activity of VER/COLL and anti-inflammatory activity of VER may represent approach to establish standard treatment of PD.

Acetylenic derivative of betulin induces apoptosis in endometrial adenocarcinoma cell line

Since betulin (Bet) and its acetylenic derivative, 28-O-propynoylbetulin (proBet) were shown to induce apoptosis in several cancer cell lines, we studied the mechanism of this process in human endometrial adenocarcinoma cells (EA). Previous studies suggested that this group of compounds affect prolidase activity (proline releasing enzyme from imidodipeptides) and collagen biosynthesis (proline utilizing process) providing substrate (proline) for proline oxidase (POX) dependent apoptosis. Here we provide evidence that Bet and proBet exhibit prolidase-inducing activity in EA cell line. However, in contrast to Bet, proBet inhibited collagen biosynthesis, increased intracellular proline concentration and induced apoptosis in EA cells, as detected by caspase-3, and -9 expressions and annexin V staining. Although POX expression was not affected by both compounds, the process of apoptosis was accompanied by increase in cytoplasmic level of proline. The mechanism for proBet-induced prolidase activity was found at the level of β1 integrin signaling. The inhibition of collagen biosynthesis was due to up-regulation of NF-κB p65, an inhibitor of collagen type I gene transcription. Although Bet and proBet induced expression of pro-apoptotic p53 in EA cells, the effect of proBet on the processes was much stronger. In contrast to proBet, Bet strongly induced expression of pro-survival factors, HIF-1α and VEGF. The data suggest that massive production of proline by proBet-dependent activation of prolidase and inhibition of proline utilization for collagen biosynthesis may represent mechanism for POX-dependent apoptosis in EA cells.

Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX–Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model

Background/Aims: The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. Methods: We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7^shPRODH/POX) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. Results: PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7^shPRODH/POX cells. All studied compounds decreased cell viability in MCF-7 and MCF-7^shPRODH/POX cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7^shPRODH/POX and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7^shPRODH/POX cells and contributed to the induction of pro-survival mode only in MCF-7^shPRODH/POX cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. Conclusion: PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7^shPRODH/POX cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways.

Effects of Novel Alkyl Pyridine Platinum Complexes on Apoptosis in Ishikawa Endometrial Cancer Cells

The aim of the present study was to examine the impact of a four platinum complexes of formula [Pt2L4(berenil)2]Cl4 where L is 3-ethylpyridine (Pt10), 3-(n-butyl)pyridine (Pt11), 4- ethylpyridine (Pt12) and 4-(t-butyl)pyridine (Pt13) on viability of Ishikawa endometrial cancer cells using the MTT assay and inhibition of [3H]thymidine incorporation into DNA. Our results confirm that compounds Pt10-Pt13 are more potent antiproliferative agents than cisplatin in endometrial cancer cells. Moreover, it was shown that all examined compounds Pt10-Pt13 inhibit collagen biosynthesis in neoplastic cells stronger than cisplatin. Flow cytometric analysis after annexin V-FITC and propidium iodide staining confirmed also that apoptosis was the main response of Ishikawa endometrial cancer cells to Pt10-Pt13 treatment. Our results suggest that apoptosis of Ishikawa endometrial cancer cell lines in the presence of Pt10-Pt13 follows the mitochondrial pathway, with the decrease in mitochondrial membrane potential and activation of caspase 9, as well as by the external pathway with the significant increase in FADD protein expression and caspase 8.

Prolidase-dependent mechanism of (Z)-8,9-epoxyheptadeca-1,11,14-triene-induced inhibition of collagen biosynthesis in cultured human skin fibroblasts

The effects of polyolefinic compound from roots of Cirsium palustre, (Z)-8,9-epoxyheptadeca-1,11,14-triene (EHT) on collagen biosynthesis, prolidase activity, expression of insulin-like growth factor receptor (IGF-IR), β1 integrin, MAP kinases (pERK1/2), the transcription factors such as nuclear factor kappa B (NF-κB) and hypoxia-inducible factor-1α (HIF-1α) were evaluated in human dermal fibroblasts treated with micromolar concentrations (40–200 μM) for 24 h. It was found that EHT-dependent inhibition of collagen biosynthesis was accompanied by parallel inhibition in prolidase activity. Since IGF-I is the most potent regulator of both processes and prolidase is regulated by β1 integrin signalling, the effect of EHT on IGF-IR and β1 integrin receptor expressions were evaluated. Exposure of the cells to EHT contributed to distinct increase in IGF-IR and slight increase in β1 integrin receptor expressions. It was accompanied by decrease in expression of pERK1/2, HIF-1α and NF-κB. EHT-dependent inhibition of collagen biosynthesis results from inhibition of prolidase activity, the enzyme involved in collagen biosynthesis.

Influence of caffeine and hyaluronic acid on collagen biosynthesis in human skin fibroblasts.

AimThe aim of this study was to evaluate the effect of caffeine on collagen biosynthesis in human skin fibroblasts and the influence of hyaluronic acid (HA) on this process.Materials and methodsCollagen, [3H]-thymidine incorporation, and prolidase activity were measured in confluent human skin fibroblast cultures that had been treated with 1, 2, and 5 mM caffeine and with caffeine and 500 μg/mL HA. Western immunoblot analysis was performed to evaluate expression of β1-integrin receptor, insulin-like growth factor receptor phospho-Akt protein and mitogen-activated protein kinase (phospho-extracellular signal-regulated kinase).ResultsCaffeine inhibited collagen biosynthesis in a dose-dependent manner. The mechanism of this process was found at the level of prolidase activity. Caffeine significantly inhibited the enzyme activity. The addition of HA had no effect on collagen biosynthesis or prolidase activity in fibroblasts incubated with caffeine. Caffeine also had an inhibitory effect on DNA biosynthesis. HA, however, did not have any significant effect on this process. The inhibition of the expression of β1-integrin and insulin-like growth factor receptor in fibroblasts incubated with the caffeine indicates a possible mechanism of inhibition of collagen biosynthesis.ConclusionCaffeine reduces collagen synthesis in human cultured skin fibroblasts. HA did not have any significant protective effect on this process. This is the first study to our knowledge that reports caffeine-induced inhibition of collagen synthesis in human skin fibroblasts.

Plasma S-nitrosothiols in systemic sclerosis and Raynaud’s phenomenon

Systemic sclerosis (SSc) is a skin disease marked by overproduction of collagen, vascular dysfunction and dysregulation of NO biosynthesis. All patients exhibit Raynaud’s phenomenon. In some cases internal organs are involved (dSSc). In limited SSc (lSSc), lesions are largely confined to skin. NO and S-nitrosothiols (RSNO) inhibit collagen biosynthesis in vitro. The study investigates levels of RSNO in patients with SSc and primary Raynauds’ phenomenon (RP). RSNO and NO 2 - were measured by chemiluminescence (NO and ozone) in plasma of patients (78% female) and controls. Skin scores show the level of disease. Statistical analyses were performed by one way ANOVA and Newman–Keuls tests. A striking reduction in the level of detectable RSNO was observed in most patients compared to controls. In healthy controls, RSNO decreased with age (above 40 years). The following levels were found in controls (6 ± 0.8 SEM, n = 26), patients with lSSc (1.31 ± 0.37, n = 34, P = 1.06E−06), dSSc (0.73 ± 0.41, n = 11, P = 00025) and RP (2.12 ± 0.57, n = 16, P = 0.002). In SSc patients, high skin scores correlated with low RSNO levels (dSSc). There were no differences in nitrite levels between groups. The results suggest a link between the availability of NO as S-nitrosothiols and the severity of SSc.

Biochemical indices in the blood of rats at different terms of reparative osteogenesis in conditions of hyperhomocysteinaemia

The effects, produced by hyperhomocysteinaemia (HHcy) and its correction with Decamevit and Glutargin on bone tissue metabolism in rats with femur fractures in different terms of reparative osteogenesis, were studied. It was established, that HHcy caused enhancement of bone tissue resorption, increase of calcium excretion with urine, inhibition of collagen biosynthesis in rats with femur fractures. HHcy induced dysregulation of reparative osteogenesis by lowering the transforming growth factor beta -1 (TGF- β1) levels in the first 2 weeks after trauma and by an excessive increase of this index on days 30-45 after the trauma. Decamevit and Glutargin reduced HHcy-induced imbalance between the processes of resorption and biosynthesis of bone tissue, and normalized the staging changes of TGF- β1 levels in blood serum of rats with femur fractures.

Inhibition of Signal Transducer and Activator of Transcription 3 (STAT3) Attenuates Interleukin-6 (IL-6)-induced Collagen Synthesis and Resultant Hypertrophy in Rat Heart

IL-6 has been shown to play a major role in collagen up-regulation process during cardiac hypertrophy, although the precise mechanism is still not known. In this study we have analyzed the mechanism by which IL-6 modulates cardiac hypertrophy. For the in vitro model, IL-6-treated cultured cardiac fibroblasts were used, whereas the in vivo cardiac hypertrophy model was generated by renal artery ligation in adult male Wistar rats (Rattus norvegicus). During induction of hypertrophy, increased phosphorylation of STAT1, STAT3, MAPK, and ERK proteins was observed both in vitro and in vivo. Treatment of fibroblasts with specific inhibitors for STAT1 (fludarabine, 50 μM), STAT3 (S31-201, 10 μM), p38 MAPK (SB203580, 10 μM), and ERK1/2 (U0126, 10 μM) resulted in down-regulation of IL-6-induced phosphorylation of specific proteins; however, only S31-201 and SB203580 inhibited collagen biosynthesis. In ligated rats in vivo, only STAT3 inhibitors resulted in significant decrease in collagen synthesis and hypertrophy markers such as atrial natriuretic factor and β-myosin heavy chain. In addition, decreased heart weight to body weight ratio and improved cardiac function as measured by echocardiography was evident in animals treated with STAT3 inhibitor or siRNA. Compared with IL-6 neutralization, more pronounced down-regulation of collagen synthesis and regression of hypertrophy was observed with STAT3 inhibition, suggesting that STAT3 is the major downstream signaling molecule and a potential therapeutic target for cardiac hypertrophy.

Scutellarin-dependent inhibition of collagen biosynthesis in cultured fibroblasts

The effects of the flavonoid compound scutellarin (SCUT) on collagen biosynthesis, prolidase activity, expression of β1 integrin, insulin-like growth factor-I (IGF-I) receptor and the transcription factor NF-κB were evaluated in human dermal fibroblasts. Confluent fibroblasts were treated with micromolar concentrations (10–30 µM) of SCUT for 24 h. It was found that a SCUT-dependent decrease in collagen biosynthesis was accompanied by an increase in prolidase activity. Since the IGF-I receptor (IGF-IR) is the most potent regulator of both collagen biosynthesis and prolidase activity, and prolidase is regulated by β1 integrin signalling, the effect of SCUT on IGF-IR and β1 integrin receptor expressions were evaluated. It was found that the exposure of the cells to SCUT contributed to an increase in IGF-IR and β1 integrin receptor expressions. This was accompanied by an increase in expression of NF-κB, the known inhibitor of collagen gene expression. These data suggest that the SCUT-dependent decrease of collagen biosynthesis in cultured human skin fibroblasts results from activation of NF-κB, which is responsible for the down-regulation of collagen gene expression.

Betulinic acid inhibits the expression of hypoxia-inducible factor 1α and vascular endothelial growth factor in human endometrial adenocarcinoma cells

Although betulinic acid (BA) is known to induce apoptosis and antiangiogenic response in tumor cells, the underlying mechanism of its action is unknown. Deregulation of tissue collagen metabolism is one of the consequences of neoplastic transformation. The final step of collagen degradation is mediated by prolidase [E.C.3.4.13.9] which may play a role in angiogenesis. The formation of new blood vessels is regulated by the hypoxia-inducible factor 1 (HIF-1). The expression of HIF-1 correlates with hypoxia-induced angiogenesis as a result of the induction of vascular endothelial cell growth factor (VEGF). Since BA evokes anticancer activity, its effect on collagen biosynthesis, HIF-1alpha and VEGF expressions, as well as prolidase activity and expression was studied in cultured endometrial adenocarcinoma (EA) cells. It was found that BA inhibits collagen biosynthesis in EA cells (5[(3)H] proline incorporation assay). It was accompanied by a parallel decrease in prolidase activity and expression and decrease in expressions of alpha(1) and alpha(2) integrins, HIF-1alpha, and VEGF (western immunoblot analysis) in cultured human EA cells. The data suggest that BA may have anti-angiogenic potential by inhibition of prolidase, HIF-1alpha and VEGF expressions, and inhibition of collagen biosynthesis.

Mechanism of betulinic acid inhibition of collagen biosynthesis in human endometrial adenocarcinoma cells

Collagen as a ligand for integrin receptors plays important role in the integrin - dependent regulation of cellular metabolism. Since betulinic acid (BA) evokes anticancer activity, its effect on collagen biosynthesis was studied in cultured endometrial adenocarcinoma cells. Confluent cells were treated with different concentrations of BA for 24 hours. It was found that BA inhibit collagen biosynthesis ([3H] proline incorporation assay). The mechanism of this phenomenon was found at the level of insulin-like growth factor-I receptor (IGF-IR) and alpha2 integrin signalling (Western immunoblot analysis). The expressions of IGF-I receptor and alpha2 integrin subunit as well as integrin activated focal adhesion kinase (FAK) were decreased in the cells treated with BA. It was accompanied by a parallel decrease in the expression of Sos protein and phosphorylated MAP-kinases (ERK1, ERK2) and up - regulation of NF-kappaB. The data suggest that BA-dependent inhibition of collagen biosynthesis in cultured human endometrial adenocarcinoma cells undergoes through alpha2 integrin and IGF-IR signaling that activate NF-kappaB, potent inhibitor of collagen gene expression.

Phosphoenolpyruvate-dependent inhibition of collagen biosynthesis, α2β1 integrin and IGF-I receptor signaling in cultured fibroblasts

The mechanism of collagen biosynthesis regulation is not fully understood. The finding that prolidase plays an important role in collagen biosynthesis and phosphoenolpyruvate inhibits prolidase activity "in vitro" led to evaluate its effect on collagen biosynthesis in cultured human skin fibroblasts. Confluent fibroblasts were treated with millimolar concentrations (1-4 mM) of phosphoenolpyruvate monopotassium salt (PEP) for 24 h. It was found that PEP-dependent decrease in prolidase activity and expression was accompanied by parallel decrease in collagen biosynthesis. However, the experiments with inhibitor of PEP production, 3-mercaptopicolinate revealed no direct correlation between collagen biosynthesis and prolidase activity and expression. Since insulin-like growth factor (IGF-I) is the most potent stimulator of both collagen biosynthesis and prolidase activity, and prolidase is regulated by beta(1) integrin signaling, the effect of PEP on IGF-I receptor (IGF-IR) and beta(1) integrin receptor expressions were evaluated. It was found that the exposure of the cells to 4 mM PEP contributed to a decrease in IGF-IR and beta(1) integrin receptor expressions. The data suggest that PEP-dependent decrease of collagen biosynthesis in cultured human skin fibroblasts may undergo through depression of alpha(2)beta(1) integrin and IGF-IR signaling. The hypothetical mechanism of the role of prolidase in IGF-IR, beta(1) integrin receptor expressions, and clinical significance of the process are discussed.

Prolidase Dependent Inhibition of Collagen Biosynthesis in Chinese Hamster Ovary Cells

Collagen is responsible for maintenance of connective tissue integrity, and through interaction with integrin receptors may participate in regulation of numerous physiological and pathological processes. An important role in collagen biosynthesis plays prolidase. It was previously found that nickel chloride inhibited prolidase activity in Chinese hamster ovary cells (CHO-C9). The cells lack any detectable ornithine aminotransferase and P5C synthase activities, and therefore require addition of free proline or glicyl-proline (converted to glycine and proline) for growth. We have found that Ni(II) contributed to decrease in collagen and hydroxyproline content in CHO cells incubated with Gly-Pro, whereas it had no effect on hydroxyproline content in the cells incubated with proline. Decrease in collagen content was not related to decrease in type I collagen mRNA level suggesting regulation of this process at post-transcriptional level. However decrease in expression of Sos and phosphorylated MAP-kinases were found in the cells growing in the presence of Gly-Pro and Ni(II). Decrease in the expression of these proteins was not related to inhibition of signalling induced by growth factors, since no changes were observed in expression of AKT in CHO cells incubated with Ni(II). The results presented provide evidence for important role of prolidase in collagen biosynthesis.