Proline-dependent regulation of collagen metabolism.

This review is focused on recent data on the role of proline (Pro) in collagen biosynthesis and cellular metabolism. It seems obvious that one of the main substrates for collagen biosynthesis Pro is required to form collagen molecule. The question raised in this review is whether the Pro for collagen biosynthesis is synthesized "de novo", comes directly from degraded proteins or it is converted from other amino acids. Recent data provided evidence that extracellular Pro (added to culture medium) had significant, but relatively little impact on collagen biosynthesis in fibroblasts (the main collagen synthesized cells) cultured in the presence of glutamine (Gln). However, extracellular Pro drastically increased collagen biosynthesis in the cells cultured in Gln-free medium. It suggests that Pro availability determines the rate of collagen biosynthesis and demand for Pro in fibroblasts is predominantly met by conversion from Gln. The potential mechanism of this process as well as possible implication of this knowledge in pharmacotherapy of connective tissue diseases is discussed in this review.

The mechanism for differential effect of nelfinavir and indinavir on collagen metabolism in human skin fibroblasts.

The mechanism for differential effects of human immune deficiency virus protease inhibitors (HIVPIs), nelfinavir (NEL) and indinavir (IND) on collagen metabolism disturbances was studied in human skin fibroblasts. It has been considered that HIVPIs-dependent deregulation of collagen biosynthesis involves prolidase (an enzyme providing proline for collagen biosynthesis), glutamine (Gln) (a substrate for proline biosynthesis), nuclear factor-κB (NF-κB) (a transcription factor that inhibit expression of type I collagen genes), ß1 integrin receptor and Akt signalling. It was found that NEL impaired collagen biosynthesis and the process was more pronounced in the presence of Gln, while IND stimulated collagen biosynthesis. NEL-dependent inhibition of collagen biosynthesis was accompanied by massive intracellular accumulation of type I collagen, while IND slightly induced this process. This effect of NEL was reversed by ascorbic acid but not N-acetylcysteine. The mechanism for the NEL-dependent defect in collagen metabolism was found at the level of prolidase activity, ß1 integrin signalling and NF-κB. NEL inhibited expression of ß1 integrin receptor, Akt and ERK1/2 and increased expression of p65 NF-κB. However, inhibitors of p65 NF-κB did not prevent NEL-dependent inhibition of collagen biosynthesis suggesting that this transcription factor is not involved in studied mechanism. Using PI3K inhibitor wortmannin that prevent phosphorylation of Akt revealed that NEL-dependent inhibition of Akt results in inhibition of collagen biosynthesis. The data suggest that differential effect of NEL and IND on collagen metabolism involves NEL-dependent down-regulation of Akt signalling and proline availability for collagen biosynthesis.

Abies Concolor Seeds and Cones as New Source of Essential Oils-Composition and Biological Activity.

The chemical composition, including the enantiomeric excess of the main terpenes, of essential oils from seeds and cones of Abies concolor was studied by chromatographic (GC) and spectroscopic methods (mass spectrometry, nuclear magnetic resonance), leading to the determination of 98 compounds. Essential oils were mainly composed of monoterpene hydrocarbons. The dominant volatiles of seed essential oil were: limonene (47 g/100 g, almost pure levorotary form) and α-pinene (40 g/100 g), while α-pinene (58 g/100 g), sabinene (11 g/100 g), and ß-pinene (4.5 g/100 g) were the predominant components of the cone oil. The seed and cone essential oils exhibited mild antibacterial activity, and the MIC ranged from 26 to 30 µL/mL against all of the tested bacterial standard strains: Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, and Klebsiella pneumoniae. The cytotoxic studies have demonstrated that tested essential oils were cytotoxic to human skin fibroblasts and human microvascular endothelial cells at concentrations much lower than the MIC. The essential oils from A. concolor seeds and cones had no toxic effect on human skin fibroblasts and human microvascular endothelial cells, when added to the cells at a low concentration (0-0.075 µL/mL) and (0-1.0 µL/mL), respectively, and cultured for 24 h.

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 ß1 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.

Exogenous proline stimulates type I collagen and HIF-1α expression and the process is attenuated by glutamine in human skin fibroblasts.

Abundance of proline (Pro) in collagen molecule led us to investigate whether Pro supply affects collagen biosynthesis in human skin fibroblasts. Treatment of the cells with milimolar concentrations (5 and 10 mM) of Pro for 24 and 48 h contributed to increase in α1 subunit of collagen type I (COL1A1) expression in both cells and culture medium. However, the effect was more pronounced in glutamine-free medium. In such condition, Pro induced collagen expression by about twofold in the cells, while in the medium only by about 30% during 24 h incubation, compared to control. In the presence of glutamine (Gln), exogenous Pro stimulated intracellular collagen expression only by about 30% during 24 h of fibroblasts incubation, and it was not accompanied by adequate increase of collagen secretion into medium. Gln alone stimulated the processes by about 2-3 fold during the course of the experiment. Pro-dependent increase in collagen expression in Gln-free medium was accompanied by increase in prolidase activity and expression of pAkt. In both Gln-free medium and Gln-supplemented medium, Pro induced expression of p53 and HIF-1α. The data suggest that availability of Gln, as a substrate for Pro biosynthesis, determine the utilization of exogenous Pro for the collagen biosynthesis.

Composition and Biological Activity of Picea pungens and Picea orientalis Seed and Cone Essential Oils.

The increasing consumption of natural products lead us to discover and study new plant materials, such as conifer seeds and cones, which could be easily available from the forest industry as a waste material, for their potential uses. The chemical composition of the essential oils of Picea pungens and Picea orientalis was fully characterized by GC and GC/MS methods. Seed and cone oils of both tree species were composed mainly of monoterpene hydrocarbons, among which limonene, α- and ß-pinene were the major, but in different proportions in the examined conifer essential oils. The levorotary form of chiral monoterpene molecules was predominant over the dextrorotary form. The composition of oils from P. pungens seeds and cones was similar, while the hydrodistilled oils of P. orientalis seeds and cones differed from each other, mainly by a higher amount of oxygenated derivatives of monoterpenes and by other higher molar mass terpenes in seed oil. The essential oils showed mild antimicrobial action, however P. orientalis cone oil exhibited stronger antimicrobial properties against tested bacterial species than those of P. pungens. Effects of the tested cone essential oils on human skin fibroblasts and microvascular endothelial cells (HMEC-1) were similar: in a concentration of 0 - 0.075 µl/ml the oils were rather safe for human skin fibroblasts and 0 - 0.005 µl/ml for HMEC-1 cells. IC50 value of Picea pungens oils was 0.115 µl/ml, while that of Picea orientalis was 0.105 µl/ml. The value of IC50 of both oils were 0.035 µl/ml for HMEC-1 cells. The strongest effect on cell viability had the oil from Picea orientalis cones, while on DNA synthesis the oil from Picea pungens cones.

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), ß1 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 ß1 integrin signalling, the effect of EHT on IGF-IR and ß1 integrin receptor expressions were evaluated. Exposure of the cells to EHT contributed to distinct increase in IGF-IR and slight increase in ß1 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.

Enalapril stimulates collagen biosynthesis through prolidase-dependent mechanism in cultured fibroblasts.

The mechanism of a lower incidence of dermatological manifestations in patients treated with enalapril compared to patients treated with other ACE-inhibitors, e.g., captopril, is not known. The finding that prolidase plays an important role in collagen biosynthesis and that some angiotensin-converting enzyme inhibitors affect prolidase activity led us to evaluate its effect on collagen biosynthesis in cultured human skin fibroblasts. Since insulin-like growth factor (IGF-I) and transforming growth factor beta 1 (TGF-ß1) are the most potent stimulators of both collagen biosynthesis and prolidase activity, and prolidase is regulated by ß1 integrin signaling, the effect of enalapril and enalaprilat on IGF-IR, TGF-ß1, and ß1 integrin receptor expressions was evaluated. Cells were treated with milimolar concentrations (0.3 and 0.5 mM) of enalapril and enalaprilat for 24 h. The activity of prolidase was determined by colorimetic assay. Collagen biosynthesis was evaluated by radiometric assay. Expression of signaling proteins was evaluated using Western blot. It was found that enalapril- and enalaprilat-dependent increase in prolidase activity and expression was accompanied by parallel increase in collagen biosynthesis. The exposure of the cells to 0.5 mM enalapril and enalaprilat contributed to increase in IGF-IR and α2ß1 integrin receptor as well as TGF-ß1 and NF-κB p65 expressions. Enalapril- and enalaprilat-dependent increase of collagen biosynthesis in fibroblasts results from increase of prolidase activity and expression, which may undergo through activation of α2ß1 integrin and IGF-IR signaling as well as upregulation of TGF-ß1 and NF-κB p65, the inhibitor of collagen gene expression.

Chemical composition and biological activity of Abies alba and A. koreana seed and cone essential oils and characterization of their seed hydrolates.

The chemical composition, including the enantiomeric excess of the main terpenes, the antimicrobial and antiradical activities, as well as the cytotoxicity of Abies alba and A. koreana seed and cone essential oils were investigated. Additionally, their seed hydrolates were characterized. In the examined oils and hydrolates, a total of 174 compounds were identified, which comprised 95.6-99.9% of the volatiles. The essential oils were mainly composed of monoterpene hydrocarbons, whereas the composition of the hydrolates, differing from the seed oils of the corresponding fir species, consisted mainly of oxygenated derivatives of sesquiterpenes. The seed and cone essential oils of both firs exhibited DPPH-radical-scavenging properties and low antibacterial activity against the bacterial strains tested. Moreover, they evoked only low cytotoxicity towards normal fibroblasts and the two cancer cell lines MCF-7 and MDA-MBA-231. At concentrations up to 50 µg/ml, all essential oils were safe in relation to normal fibroblasts. Although they induced cytotoxicity towards the cancer cells at concentrations slightly lower than those required for the inhibition of fibroblast proliferation, their influence on cancer cells was weak, with IC50 values similar to those observed towards normal fibroblasts.

The mechanism of oxythiamine-induced collagen biosynthesis in cultured fibroblasts.

The oxythiamine (OXY) is antivitamin of thiamine. The finding that OXY increases the cytoplasmic concentration of pyruvate, known to enhance collagen biosynthesis, led us to investigate the mechanism of this antivitamin action on collagen biosynthesis in cultured human skin fibroblasts. Confluent fibroblasts were treated with micromolar concentrations (30-1,000 µM) of OXY for 24 and 48 h. It was found that OXY-dependent increase in collagen biosynthesis was accompanied by parallel increase in prolidase activity and level, compared to untreated cells. Since phosphoenolpyruvate (PEP) is known as an inhibitor of prolidase-the enzyme that plays important role in collagen biosynthesis, the mechanism of pyruvate interconversion was considered as a regulatory switch in collagen biosynthesis. In fact, 3-MPA, specific inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), contributed to up-regulation of prolidase activity, suggesting that down-regulation of PEP formation is an underlying mechanism. Since collagen biosynthesis and prolidase activity are regulated by signal induced by activated α2ß1 integrin receptor as well as insulin-like growth factor-I receptor (IGF-IR), the expression of these receptors was measured by Western immunoblot analysis. The exposure of the cells to OXY contributed to decrease in IGF-IR, α2ß1 integrin receptor, pERK1/2, and NF-κB p65 expressions. It was accompanied by increase in total ERK1/2 expression and induction of phosphorylation of Akt protein. The data suggest that OXY-dependent increase of collagen biosynthesis in cultured human skin fibroblasts results from activation of prolidase activity and level, induction in pAkt expression and down-regulation of pERK1/2 and NF-κB p65, the known inhibitor of collagen gene expression.

UVC inhibits collagen biosynthesis through up-regulation of NF-κB p65 signaling in cultured fibroblasts.

The effects of UVC on collagen biosynthesis, prolidase activity, expression of α2ß1 integrin, IGF-I receptor, FAK, MAP-kinases (ERK1 and ERK2) and the transcription factor NF-κB p65 were evaluated in human dermal fibroblasts. Confluent fibroblasts were treated with UVC light at a rates of 30 and 60 J/m(2). It was found that UVC-dependent decrease in collagen biosynthesis was not accompanied by parallel decrease in prolidase activity and expression. Since insulin-like growth factor receptor (IGF-IR) and α2ß1 integrin signaling are the most potent regulators of collagen biosynthesis, the effect of UVC on IGF-IR and α2ß1 integrin receptor expressions were evaluated. It was found that the exposure of the cells to UVC contributed to decrease in α2ß1 integrin receptor and FAK expression and to an increase in IGF-IR and pERK1, pERK2 expressions. It was accompanied by an increase in the expression of NF-κB p65, the known inhibitor of collagen gene expression. The data suggest that UVC-dependent decrease of collagen biosynthesis in cultured human skin fibroblasts results from decrease in α2ß1 integrin receptor signaling and activation of NF-κB p65, that is responsible for down-regulation of collagen gene expression.

The mechanism of hydralazine-induced collagen biosynthesis in cultured fibroblasts.

The finding that hydralazine (HYD) affects collagen metabolism led us to investigate the mechanism of its action on collagen biosynthesis, prolidase expression and activity, expression of α2ß1 integrin, insulin-like growth factor-I receptor (IGF-IR), focal adhesion kinase (FAK), mitogen-activated protein (MAP) kinases (ERK1, ERK2), and transcription factors hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-κB p65 (NF-κB p65) in human dermal fibroblasts. Confluent fibroblasts were treated with micromolar concentrations (50-500 µM) of HYD for 24 h. HYD had no influence on cell viability. It was found that HYD-dependent increase in collagen biosynthesis was accompanied by a parallel increase in prolidase activity and expression, HIF-1α expression, and decrease in DNA biosynthesis, compared to untreated cells. Since collagen biosynthesis and prolidase activity are regulated by a signal induced by activated α2ß1 integrin receptor as well as IGF-IR, the expression of these receptors was measured by Western immunoblot analysis. The exposure of the cells to HYD contributed to the increase in IGF-IR expression without any effect on α2ß1 integrin receptor and FAK expressions. It was accompanied by a decrease in expression of MAP kinases and NF-κB p65, the known inhibitor of collagen gene expression. The data suggest that the HYD-dependent increase of collagen biosynthesis in cultured human skin fibroblasts results from activation of IGF-IR expression and prolidase activity and downregulation of NF-κB p65.

The chemical composition of the essential oils of Cirsium palustre and C. rivulare and their antiproliferative effect.

The composition of the essential oils of Cirsium palustre and C. rivulare and their antiproliferative activity against breast adenocarcinoma cells (MCF-7 and MDA-MBA-231) were investigated. The essential oils obtained by hydro-distillation from the roots (yield 0.2 and 0.1% v/w, respectively), leaves and inflorescences (yield below 0.01%), were analyzed by gas chromatography coupled with mass spectrometry (GC-MS). The composition of the essential oils in the respective organs of each plant differed considerably. On the other hand, similarities were observed in the composition of root and leaf oils. In C. palustre and C. rivulare root oil, 50 and 39 constituents were identified, accounting for 95.3% and 92.4% of the total content. The main components were aplotaxene and its derivatives, representing 78.6% and 46.6% of the total content. In leaf oils of both species, 59 and 49 compounds, respectively, were identified, representing 67.4% and 78.3% of the total content. The major constituents were beta-damascenone (4.1% and 13.4%, respectively) and beta-ionone (6.7% and 5.3%, respectively). Short-chain saturated and unsaturated aliphatic alcohols and aldehydes constituted another important group of compounds (17.7% and 9.0%, respectively). The essential oils of the roots have moderate anti-proliferative activity, with IC50 values ranging from 110 to 140 microg/mL. These concentrations were below the level able to inhibit the proliferation of healthy cells.

Thrombin-dependent modulation of ß1-integrin-mediated signaling up-regulates prolidase and HIF-1α through p-FAK in colorectal cancer cells.

Products of prolidase [E.C. 3.4.13.9] activity, proline or hydroxyproline, contribute to up-regulation of hypoxia-inducible factor-1α (HIF-1α). Prolidase activity is regulated by ß(1)-integrin signaling. We studied the effects of echistatin (a well-known disintegrin) and thrombin (a serine protease capable of activation of integrin α(2)ß(1) receptor) on prolidase activity and expressions of prolidase, α(2)ß(1)-integrin receptor, focal adhesion kinase (FAK), MAP-kinases (ERK(1) and ERK(2)), and nuclear HIF-1α in human colon adenocarcinoma (DLD-1) cells. It has been found that treatment of the cells with thrombin contributes to decrease in the expression of prolidase and simultaneously increase in its phosphorylation, resulting in maintenance of the enzyme activity. The phenomenon was accompanied by thrombin-dependent recovery of depressed autophosphorylation of FAK (pY(397)) under the effect of FAK inhibitor (1,2,4,5-benzenetetramine tetrahydrochloride). Although integrin α(2)ß(1) receptor expression was not affected by thrombin, the signaling induced by thrombin up-regulated nuclear HIF-1α expression. It was accompanied by increase in the expression of MAP kinases, ERK1 and ERK2. It suggests that integrin-dependent signaling through p-FAK is up-regulated in DLD-1 cells and it may represent potential target for anti-cancer therapy.

Scutellarin-dependent inhibition of collagen biosynthesis in cultured fibroblasts.

The effects of the flavonoid compound scutellarin (SCUT) on collagen biosynthesis, prolidase activity, expression of ß1 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 ß1 integrin signalling, the effect of SCUT on IGF-IR and ß1 integrin receptor expressions were evaluated. It was found that the exposure of the cells to SCUT contributed to an increase in IGF-IR and ß1 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 1alpha 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.

In vitro antiproliferative and antifungal activity of essential oils from Erigeron acris L. and Erigeron annuus (L.) Pers.

Antiproliferative and antifungal activities of essential oils from Erigeron acris root and herb and from Erigeron annuus herb were investigated. The cell viability assay was performed in cultured fibroblasts, cancer cell lines (MCF-7 and MDA-MBA-231), and endometrial adenocarcinoma (Ishikawa) cells as well as colon adenocarcinoma (DLD-1) cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The essential oil from E. acris root showed the highest antiproliferative activity in the MCF-7 cell line with an IC50 value of 14.5 microg/mL. No effect of the essential oil on normal cells at that concentration was found. Antifungal activity against various strains of five Candida species, i.e. C. albicans, C. glabrata, C. tropicalis, C. krusei, and C. parapsilosis, was tested by the microdilution method. It was found that all examined oils can be useful as antifungal agents against the above-mentioned species, but the essential oil of E. acris herb was the most active. Their minimum inhibitory concentrations (MIC) ranged from 30 to 0.4 microL/mL. The data presented suggest that essential oils from E. acris and E. annuus possess antifungal activity against Candida spp. and antiproliferative activity against breast cancer MCF-7 cells.

The mechanism of butyrate-induced collagen biosynthesis in cultured fibroblasts.

The data showing that butyrate may play an important role in cellular metabolism led us to study its effect on collagen biosynthesis in cultured fibroblasts. Since insulin-like growth factor-I (IGF-I) is the most potent stimulator of collagen biosynthesis in fibroblasts, the effect of butyrate on IGF-I receptor (IGF-IR) expression was evaluated. Confluent human dermal fibroblasts were treated with millimolar concentrations of sodium butyrate (NaB) for 48 hours. It was found that butyrate induced collagen biosynthesis and prolidase activity. It was found that the exposure of the cells to 4 mM butyrate contributed to a distinct increase in IGF-IR. It was accompanied by a parallel increase in the expression of SOS protein and MAP-kinases (ERK1, ERK2). It was found that the MEK inhibitor decreased collagen biosynthesis and expression of MAP-kinases (ERK1, ERK2), while NaB counteracted the process. The data suggest that butyrate-dependent stimulation of collagen biosynthesis in cultured human skin fibroblasts undergoes through IGF-IR signaling.

The mechanism of butyrate-induced collagen biosynthesis in cultured fibroblasts.

The data showing that butyrate may play an important role in cellular metabolism led us to study its effect on collagen biosynthesis in cultured fibroblasts. Since insulin-like growth factor-I (IGF-I) is the most potent stimulator of collagen biosynthesis in fibroblasts, the effect of butyrate on IGF-I receptor (IGF-IR) expression was evaluated. Confluent human dermal fibroblasts were treated with millimolar concentrations of sodium butyrate (NaB) for 48 hours. It was found that butyrate induced collagen biosynthesis and prolidase activity. It was found that the exposure of the cells to 4 mM butyrate contributed to a distinct increase in IGF-IR. It was accompanied by a parallel increase in the expression of Sos protein and MAP-kinases (ERK1, ERK2). It was found that the MEK inhibitor decreased collagen biosynthesis and expression of MAP-kinases (ERK1, ERK2), while NaB counteracted the process. The data suggests that butyrate-dependent stimulation of collagen biosynthesis in cultured human skin fibroblasts undergoes through IGF-IR signaling.

Phosphoenolpyruvate-dependent inhibition of collagen biosynthesis, alpha2beta1 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.