Protein Expression In Fibroblasts Research Articles

Transient Receptor Potential Canonical-3 Channel–Dependent Fibroblast Regulation in Atrial Fibrillation

Fibroblast proliferation and differentiation are central in atrial fibrillation (AF)-promoting remodeling. Here, we investigated fibroblast regulation by Ca(2+)-permeable transient receptor potential canonical-3 (TRPC3) channels. Freshly isolated rat cardiac fibroblasts abundantly expressed TRPC3 and had appreciable nonselective cation currents (I(NSC)) sensitive to a selective TPRC3 channel blocker, pyrazole-3 (3 μmol/L). Pyrazole-3 suppressed angiotensin II-induced Ca(2+) influx, proliferation, and α-smooth muscle actin protein expression in fibroblasts. Ca(2+) removal and TRPC3 blockade suppressed extracellular signal-regulated kinase phosphorylation, and extracellular signal-regulated kinase phosphorylation inhibition reduced fibroblast proliferation. TRPC3 expression was upregulated in atria from AF patients, goats with electrically maintained AF, and dogs with tachypacing-induced heart failure. TRPC3 knockdown (based on short hairpin RNA [shRNA]) decreased canine atrial fibroblast proliferation. In left atrial fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial tachypacing, TRPC3 protein expression, currents, extracellular signal-regulated kinase phosphorylation, and extracellular matrix gene expression were all significantly increased. In cultured left atrial fibroblasts from AF dogs, proliferation rates, α-smooth muscle actin expression, and extracellular signal-regulated kinase phosphorylation were increased and were suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF atria; experimental microRNA-26 knockdown reproduced AF-induced TRPC3 upregulation and fibroblast activation. MicroRNA-26 has NFAT (nuclear factor of activated T cells) binding sites in the 5' promoter region. NFAT activation increased in AF fibroblasts, and NFAT negatively regulated microRNA-26 transcription. In vivo pyrazole-3 administration suppressed AF while decreasing fibroblast proliferation and extracellular matrix gene expression. TRPC3 channels regulate cardiac fibroblast proliferation and differentiation, likely by controlling the Ca(2+) influx that activates extracellular signal-regulated kinase signaling. AF increases TRPC3 channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo, TRPC3 blockade prevents AF substrate development in a dog model of electrically maintained AF. TRPC3 likely plays an important role in AF by promoting fibroblast pathophysiology and is a novel potential therapeutic target.

Topical treatment with anti-oxidants and Au nanoparticles promote healing of diabetic wound through receptor for advance glycation end-products

Impairment in diabetic wound healing constitutes an enormous biomedical burden. The receptor for advanced glycation end-products (RAGE) expression in the diabetic cutaneous wound may play a key role. However, the relationship between RAGE expression and topical application of anti-oxidant agents with gold nanoparticles (AuNP) in cutaneous diabetic wounds remains unclear. We tested the 3–5nm AuNP, epigallocatechin gallate (EGCG), and α-lipoic acid (ALA) could change the RAGE expression and be helpful in diabetic wound. The mixture of AuNP+EGCG+ALA (AuEA) significantly attenuated the AGE-induced RAGE protein expression in fibroblasts (Hs68). Topical EGCG+ALA (EA) and AuEA application accelerated wound healing on diabetic mouse skin and decreased the RAGE expression. Vascular endothelial growth factor but not angiopoietin-1 significantly increased after EA or AuEA treatment for 7days. Angiopoietin-2 significantly decreased at day 7 in AuEA group. Furthermore, immunoblotting of diabetic wound tissue showed significant decrease of CD68 expression from day 3 to day 7. The results suggest that combination of AuNP, EGCG, and ALA significantly accelerated diabetic cutaneous wound healing through angiogenesis regulation and anti-inflammatory effects. Blockade of RAGE by anti-oxidant agents and nanoparticles may restore effective wound healing in diabetic ulcer.

Cysteinyl leukotrienes regulate TGF-β1 and collagen production by bronchial fibroblasts obtained from asthmatic subjects

Cysteinyl leukotrienes (CysLTs) play an important role in airway inflammation in asthma but their role in airway remodeling is not completely known. CysLTs receptors and procollagen I(α(1)) mRNA were determined by qPCR. Procollagen protein production was measured by RIA and TGF-β(1) expression was determined by ELISA. TGF-β receptor expression was assessed by western blots. CysLT1R, TGF-β-R1 and active TGF-β(1) are highly expressed in cells from asthmatics compared to normal controls. LTD(4) increased significantly procollagen I(α(1)) mRNA and protein expression in fibroblasts from asthmatics. This increase was blocked by CysLTs receptor antagonist. LTD(4) increased significantly mRNA expression of TGF-β(1) and active form production in fibroblasts from asthmatics. Inhibition of TGF-β(1) signaling blocked LTD(4)-induced procollagen I(α(1)) expression. Fibroblasts from asthmatic subjects express high level of CysLT1R. LTD(4) regulates procollagen I(α(1)) transcription in fibroblasts derived from asthmatic patients by modulating TGF-β(1) expression. This suggests that CysLTs may play a role in regulating collagen deposition in asthma.

A proteomic analysis of the functional effects of fatty acids in NIH 3T3 fibroblasts.

Previous studies have demonstrated that long chain fatty acids influence fibroblast function at sub-lethal concentrations. This study is the first to assess the effects of oleic, linoleic or palmitic acids on protein expression of fibroblasts, as determined by standard proteomic techniques. The fatty acids were not cytotoxic at the concentration used in this work as assessed by membrane integrity, DNA fragmentation and the MTT assay but significantly increased cell proliferation. Subsequently, a proteomic analysis was performed using two dimensional difference gel electrophoresis (2D-DIGE) and MS based identification. Cells treated with 50 μM oleic, linoleic or palmitic acid for 24 h were associated with 24, 22, 16 spots differentially expressed, respectively. Among the identified proteins, α-enolase and far upstream element binding protein 1 (FBP-1) are of importance due to their function in fibroblast-associated diseases. However, modulation of α-enolase and FBP-1 expression by fatty acids was not validated by the Western blot technique.

A novel cell permeant peptide inhibitor of MAPKAP kinase II inhibits intimal hyperplasia in a human saphenous vein organ culture model

The present study was aimed at developing a new cell-permeant peptide inhibitor (MK2i) of the kinase that phosphorylates and activates heat-shock protein (HSP)27 (MAPKAP kinase II), and evaluating the ability of this peptide to inhibit HSP27 phosphorylation and intimal thickening. The ability of MK2i to reduce HSP27 phosphorylation and cell migration was evaluated in A7R5 cells stimulated with arsenite or lysophosphatidic acid. Stable isotopic labeling using amino acids in cell culture, in combination with liquid chromatography mass spectrometry, was used to characterize the effect of MK2i on global protein expression in fibroblasts. The effect of MK2i on intimal thickening and connective tissue growth factor expression was evaluated in human saphenous vein (HSV) rings maintained with 30% fetal bovine serum for 14 days by light microscopy and immunoblotting. Pretreatment of cells with MK2i (10 μM) prior to arsenite or lysophosphatidic acid stimulation decreased phosphorylation of HSP27 (36% ± 9% and 33% ± 10%, respectively) compared with control (not pretreated) cells. MK2i also inhibited A7R5 migration, and downregulated the transforming growth factor-induced expression of collagen and fibronectin in keloid cells, two major matrix proteins involved in the development of intimal hyperplasia. Treatment of HSV segments with MK2i enhanced relaxation, reduced HSP27 phosphorylation (40% ± 17%), connective tissue growth factor expression (17% ± 5%), and intimal thickness (48.2% ± 10.5%) compared with untreated segments. On the other hand, treatment with a recombinant fusion protein containing a cell-permeant peptide attached to the HSP27 sequence increased intimal thickness of HSV segments by 48% ± 14%. Our results suggest that HSP27 may play a role in the development of processes leading to intimal hyperplasia in HSV, and reduction of HSP27 phosphorylation by MK2i may be a potential strategy to inhibit the development of intimal hyperplasia in HSV to prevent the autologous vascular graft failure.

The splicing regulator Sam68 binds to a novel exonic splicing silencer and functions in SMN2 alternative splicing in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. An almost identical SMN2 gene is unable to compensate for this deficiency because a single C-to-T transition at position +6 in exon-7 causes skipping of the exon by a mechanism not yet fully elucidated. We observed that the C-to-T transition in SMN2 creates a putative binding site for the RNA-binding protein Sam68. RNA pull-down assays and UV-crosslink experiments showed that Sam68 binds to this sequence. In vivo splicing assays showed that Sam68 triggers SMN2 exon-7 skipping. Moreover, mutations in the Sam68-binding site of SMN2 or in the RNA-binding domain of Sam68 completely abrogated its effect on exon-7 skipping. Retroviral infection of dominant-negative mutants of Sam68 that interfere with its RNA-binding activity, or with its binding to the splicing repressor hnRNP A1, enhanced exon-7 inclusion in endogenous SMN2 and rescued SMN protein expression in fibroblasts of SMA patients. Our results thus indicate that Sam68 is a novel crucial regulator of SMN2 splicing.

Keratinocyte-releasable factors increased the expression of MMP1 and MMP3 in co-cultured fibroblasts under both 2D and 3D culture conditions

Matrix metalloproteinases (MMPs) are key elements in extracellular matrix (ECM) degradation and scar remodeling during the wound-healing process. Our previous data revealed that keratinocyte-releasable factors significantly increased the expression of fibroblast MMPs in monolayer-cultured fibroblasts. In this study, we analyzed the differences in the MMP expressions of fibroblasts in a three-dimensional fibroblast-populated collagen gel (3D FPCG) from that in a two-dimensional monolayer-cultured fibroblasts when both co-cultured with keratinocytes. Differential mRNA and protein expression of fibroblasts were examined by microarray, RT-PCR, and western blot. Our results showed that fibroblasts co-cultured with keratinocytes in a 3D FPCG expressed significantly higher MMP1 and MMP3 at the gene and protein levels. Due to the physiological advantages of a 3D FPCG model to a 2D system, we concluded that the 3D FPCG model may provide a better means of understanding the fibroblast-keratinocyte cross-talk during the wound-healing process.

Targeting Focal Adhesion Kinase With Small Interfering RNA Prevents and Reverses Load-Induced Cardiac Hypertrophy in Mice

Hypertrophy is a critical event in the onset of failure in chronically overloaded hearts. Focal adhesion kinase (FAK) has attracted particular attention as a mediator of hypertrophy induced by increased load. Here, we demonstrate increased expression and phosphorylation of FAK in the hypertrophic left ventricles (LVs) of aortic-banded mice. We used an RNA interference strategy to examine whether FAK signaling plays a role in the pathophysiology of load-induced LV hypertrophy and failure. Intrajugular delivery of specific small interfering RNA induced prolonged FAK silencing ( approximately 70%) in both normal and hypertrophic LVs. Myocardial FAK silencing was accompanied by prevention, as well as reversal, of load-induced left ventricular hypertrophy. The function of LVs was preserved and the survival rate was higher in banded mice treated with small interfering RNA targeted to FAK, despite the persistent pressure overload. Studies in cardiac myocytes and fibroblasts harvested from LVs confirmed the ability of the systemically administered specific small interfering RNA to silence FAK in both cell types. Further analysis indicated attenuation of cardiac myocyte hypertrophic growth and of the rise in the expression of beta-myosin heavy chain in overloaded LVs. Moreover, FAK silencing was demonstrated to attenuate the rise in the fibrosis, collagen content, and activity of matrix metalloproteinase-2 in overloaded LVs, as well as the rise of matrix metalloproteinase-2 protein expression in fibroblasts harvested from overloaded LVs. This study provides novel evidence that FAK may be involved in multiple aspects of the pathophysiology of cardiac hypertrophy and failure induced by pressure overload.

The role of fibroblasts and fibroblast‐derived factors in periprosthetic osteolysis

This study was undertaken to investigate how fibroblasts respond to stimulation with particulate wear debris and/or conditioned media obtained from pathologic tissue, and whether these activated fibroblasts express compounds that are involved in bone resorption. Conditioned media from explant cultures of synovial tissue, periprosthetic soft tissue (interface membranes), titanium particles, and proinflammatory cytokines were used to stimulate fibroblasts. RNase protection assay was used to measure altered gene expression, and enzyme-linked immunosorbent assay, Western blot hybridization, and flow cytometry were used to determine fibroblast protein expression. Tartrate-resistant acid phosphatase staining was used to identify multinucleated osteoclast-like cells. The most dominant compounds measured in the conditioned media from interface membranes were tumor necrosis factor alpha (TNFalpha), monocyte chemoattractant protein 1 (MCP-1), interleukin-1beta (IL-1beta), IL-6, IL-8, and vascular endothelial growth factor. Fibroblasts phagocytosed particulate wear debris and responded to cytokine/chemokine stimulation. The most prominent up-regulated genes and proteins secreted by fibroblasts in response to stimulation were matrix metalloproteinase 1, MCP-1, IL-1beta, IL-6, IL-8, cyclooxygenase 1 (COX-1), COX-2, leukemia inhibitory factor 1, transforming growth factor beta1 (TGFbeta1), and TGFbeta receptor type I. In addition, interface membrane fibroblasts expressed RANKL and osteoprotegerin in response to stimulation with conditioned media, TNFalpha, or IL-1beta. Stimulated fibroblasts cocultured with bone marrow cells in the presence of macrophage colony-stimulating factor induced osteoclastogenesis. Interface membrane fibroblasts respond directly to particulate wear debris, possibly via phagocytosis, expressing proinflammatory cytokines and RANKL. Thus, these cells may be actively involved in osteoclastogenesis and pathologic (periprosthetic) bone resorption.

Long-Chain Polyunsaturated Fatty Acids Upregulate LDL Receptor Protein Expression in Fibroblasts and HepG2 Cells

The objective of this study was to investigate the effect of individual PUFAs on LDL receptor (LDLr) expression in human fibroblasts and HepG2 cells, and to evaluate whether acyl CoA:cholesterol acyltransferase (ACAT) and sterol regulatory element-binding protein 1 (SREBP-1) were involved in the regulation of LDLr expression by fatty acids. When fibroblasts and HepG2 cells were cultured with serum-free defined medium for 48 h, there was a 3- to 5-fold (P < 0.05) increase in LDLr protein and mRNA levels. Incubation of fibroblasts and HepG2 cells in serum-free medium supplemented with 25-hydroxycholesterol (25OH-cholesterol, 5 mg/L) for 24 h decreased LDLr protein and mRNA levels by 50-90% (P < 0.05). Arachidonic acid [AA, 20:4(n-6)], EPA [20:5(n-3)], and DHA [22:6(n-3)] antagonized the depression of LDLr gene expression by 25OH-cholesterol and increased LDLr protein abundance 1- to 3-fold (P < 0.05), but had no significant effects on LDLr mRNA levels. Oleic (18:1), linoleic (18:2), and alpha-linolenic acids [18:3(n-3)] did not significantly affect LDLr expression. ACAT inhibitor (58-035, 1 mg/L) attenuated the regulatory effect of AA on LDLr protein abundance by approximately 40% (P < 0.05), but did not modify the regulatory effects of other unsaturated fatty acids in HepG2 cells. The present results suggest that AA, EPA, and DHA increase LDLr protein levels, and that ACAT plays a role in modulating the effects of AA on LDLr protein levels. Furthermore, the effects of the fatty acids appeared to be independent of any change in SREBP-1 protein.

Proteomic Analysis of Protein Components in Periodontal Ligament Fibroblasts

Characterization of periodontal ligament (PDL) fibroblast proteome is an important tool for understanding PDL physiology and regulation and for identifying disease-related protein markers. PDL fibroblast protein expression has been studied using immunological methods, although limited to previously identified proteins for which specific antibodies are available. We applied proteomic analysis coupled with mass spectrometry and database knowledge to human PDL fibroblasts. We detected 900 spots and identified 117 protein spots originating in 74 different genes. In addition to scaffold cytoskeletal proteins, e.g., actin, tubulin, and vimentin, we identified proteins implicated with cellular motility and membrane trafficking, chaparonine, stress and folding proteins, metabolic enzymes, proteins associated with detoxification and membrane activity, biodegradative metabolism, translation and transduction, extracellular proteins, and cell cycle regulation proteins. Most of these identified proteins are closely related to the extensive PDL fibroblasts' functions and homeostasis. Our PDL fibroblast proteome map can serve as a reference map for future clinical studies as well as basic research.

Peroxisome Proliferator-activated Receptor γ-mediated Transcriptional Up-regulation of the Hepatocyte Growth Factor Gene Promoter via a Novel Composite cis-Acting Element

Hepatocyte growth factor (HGF) is a pleotropic polypeptide that can function as a morphogen, motogen, mitogen, angiogen, carcinogen, and tumor suppressor, depending on the target cell and tissue. Previous studies from our laboratory using transgenic mice have shown that HGF gene expression is tightly regulated at the transcriptional level and that the upstream regulatory elements are crucial for the control of HGF gene transcription. In the present study, we have identified and characterized one of these elements as a peroxisome proliferator-activated receptor gamma (PPARgamma)-responsive element. This regulatory element was localized at -246 to -233 base pairs upstream from the transcription start site of the HGF gene promoter having the sequence GGGCCAGGTGACCT. Gel mobility shift and supershift assays demonstrated that this cis-acting element strongly binds to the PPARgamma isoforms as well as to chicken ovalbumin upstream promoter-transcription factor, a member of the orphan nuclear receptor subfamily. Mutational analysis and gel mobility band shift assays indicated that the binding site is an inverted repeat of the AGGTCA motif with two spacers (inverted repeat 2 configuration) and that the two spacers are important for PPARgamma binding. This binding site overlaps with functional binding sites for activating protein-2, nuclear factor 1, and upstream stimulatory factor, and together, they constitute a multifunctional composite binding site through which these different transcription factors exert their regulatory effects on HGF promoter activity. Functional assays revealed that PPARgamma, with its ligand, 15-deoxy-prostaglandin J2, strongly stimulates HGF promoter activity. On the other hand, nuclear factor 1, activating protein-2, and chicken ovalbumin upstream promoter-transcription factor transcription factors repress the stimulatory action of PPARgamma by competing with PPARgamma for their overlapping binding sites. Furthermore, for the first time, our studies demonstrate that the PPARgamma ligand, 15-deoxy-prostaglandin J2, induces endogenous HGF mRNA and protein expression in fibroblasts in culture.

Autocrine Overexpression of CTGF Maintains Fibrosis: RDA Analysis of Fibrosis Genes in Systemic Sclerosis

We have used representational difference analysis (RDA) to identify up-regulated genes in skin fibroblasts from fibrotic lesions obtained from patients with systemic sclerosis (scleroderma). RDA of cDNA libraries derived from fibroblasts from involved and uninvolved skin detected several differentially expressed genes. One such gene consistently up-regulated in scleroderma cells coded for human connective tissue growth factor (CTGF). Other studies described here show that the CTGF protein is readily detected in cultures of systemic sclerosis fibroblasts but was not detected in comparable normal cells. High levels of CTGF are also evident in biological fluids from patients with systemic sclerosis. TGFβ stimulates CTGF production in both normal and systemic sclerosis fibroblasts with the latter found to be higher producers. Moreover, an analysis of constitutive and TGFβ-induced CTGF gene activation showed altered and elevated transcriptional responses in systemic sclerosis cells compared with controls. CTGF stimulated a two- to threefold increase in proα1(I) collagen and fibronectin synthesis by both dermal and lung fibroblasts in culture and promoted significant matrix remodeling of fibroblast-populated three-dimensional collagen lattices. A direct relation between the overexpression of CTGF and elevated collagen synthesis was suggested by the observation that transfection of a CMV–CTGF cDNA construct and protein expression in fibroblasts increased the transcription of a Col 1α2 promoter–reporter construct to levels seen in systemic sclerosis fibroblasts. Using Col 1α2 promoter deletion constructs the CTGF responsive element was localized to the first 379 bp upstream of the transcriptional start site. These data indicate that there is an overexpression of CTGF in the systemic sclerosis cells, probably due to increased gene transcription, and suggest that the dysregulation of CTGF production is an important factor in fibroblast activation and the excessive deposition of collagen in systemic sclerosis.

Kinesin-like protein CENP-E is upregulated in rheumatoid synovial fibroblasts.

Our aim was to identify specifically expressed genes using RNA arbitrarily primed (RAP)-polymerase chain reaction (PCR) for differential display in patients with rheumatoid arthritis (RA). In RA, amplification of a distinct PCR product suitable for sequencing could be observed. Sequence analysis identified the PCR product as highly homologous to a 434 base pair segment of the human centromere kinesin-like protein CENP-E. Differential expression of CENP-E was confirmed by quantitative reverse transcription PCR, immunohistochemistry and in situ hybridization. CENP-E expression was independent from prednisolone and could not be completely inhibited by serum starvation. RAP-PCR is a suitable method to identify differentially expressed genes in rheumatoid synovial fibroblasts. Also, because motifs of CENP-E show homologies to jun and fos oncogene products and are involved in virus assembly, CENP-E may be involved in the pathophysiology of RA.

Characteristics of growth, morphology, contractility, and protein expression of fibroblasts derived from keloid

Phenotypic alterations of keloid-derived fibroblasts were characterized by comparison with the phenotypes of normal fibroblasts from the same patient. Explant cultures of keloids showed unique features. Keloid explants contracted considerably and reduced their size during culture, whereas the size of normal skin explants remained unchanged. Enlarged cells were found among fibroblasts which had grown out of all the explants and were morphologically distinct from fibroblasts; however, keloid explants produced many more of them than did the normal tissues. The growth rate of fibroblast colonies formed from normal explants was five times higher than keloid explants. Keloid fibroblasts which had been serially cultivated contracted lattices of collagen gels at a rate similar to normal fibroblasts. Proteins extracted from serially cultivated fibroblasts were mapped on polyacrylamide two-dimensional electrophoretic gels. No significant qualitative alterations in protein expression in keloid cells were found as compared with normal fibroblasts. However, some quantitative changes were found between the two. A computer-assisted image analyzer detected 151 polypeptide spots--50 spots (33%) of which increased their amounts in keloid cells, whereas 34 spots (22.5%) decreased in comparison with normal fibroblasts. Sixteen major polypeptides were identified as known proteins with the aid of time-of-flight mass spectrometry. The level of expression of these identified proteins was similar between normal and keloid cells, except stathmin whose expression was suppressed in keloid fibroblasts.

Effect of glucocorticoid on insulin-like growth factor (IGF) regulation of IGF-binding protein expression in fibroblasts.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) can determine IGF biological competence at the cellular level. IGF-I itself has been shown to be an important peptide regulator of local IGFBP availability. Glucocorticoid also has major effects on IGFBP expression. In the present study, we assessed integrated IGF-I and glucocorticoid regulation of IGFBP messenger RNA (mRNA) and protein expression in two fibroblast model systems. In bovine fibroblasts, IGF-I treatment induced IGFBP-3 and IGFBP-5 mRNA and protein secretion, and had a moderate effect on IGFBP-4 expression. Dexamethasone had little effect on the IGF-induced increase in IGFBP-3, but completely blocked the increase in IGFBP-5 expression. Basal IGFBP-4 expression was inhibited by dexamethasone, and this effect was counteracted by IGF-I. IGFBP-2 expression did not vary with IGF-I or dexamethasone treatment in these cells; IGFBP-1 mRNA was not detectable, and IGFBP-6 mRNA was low and inconsistent. In human fibroblasts, IGF-I treatment increased levels of IGFBP-3 and decreased levels of IGFBP-4 without influencing mRNA expression. IGF-I also increased steady state levels of IGFBP-5 mRNA. Dexamethasone alone decreased IGFBP-3, IGFBP-4, and IGFBP-5 mRNA, but it had no significant effect on IGFBP-3, -4, or -5 expression in the presence of IGF-I. Human fibroblast IGFBP-6 expression was stable under the different culture conditions; IGFBP-1 and -2 mRNA were not detected. These data demonstrate that IGF peptide and glucocorticoid individually modulate IGFBP expression and indicate that glucocorticoid has distinct effects on IGF regulation of IGFBP depending upon the particular IGFBP and the underlying mechanism of IGF regulation. Bovine fibroblasts may provide a useful model system to probe the molecular mechanisms of IGFBP-3, -4, and -5 gene expression and regulation by IGF-I and glucocorticoid, whereas human fibroblasts may be suitable for studying posttranscriptional interactions.

Effect of glucocorticoid on insulin-like growth factor (IGF) regulation of IGF-binding protein expression in fibroblasts

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) can determine IGF biological competence at the cellular level. IGF-I itself has been shown to be an important peptide regulator of local IGFBP availability. Glucocorticoid also has major effects on IGFBP expression. In the present study, we assessed integrated IGF-I and glucocorticoid regulation of IGFBP messenger RNA (mRNA) and protein expression in two fibroblast model systems. In bovine fibroblasts, IGF-I treatment induced IGFBP-3 and IGFBP-5 mRNA and protein secretion, and had a moderate effect on IGFBP-4 expression. Dexamethasone had little effect on the IGF-induced increase in IGFBP-3, but completely blocked the increase in IGFBP-5 expression. Basal IGFBP-4 expression was inhibited by dexamethasone, and this effect was counteracted by IGF-I. IGFBP-2 expression did not vary with IGF-I or dexamethasone treatment in these cells; IGFBP-1 mRNA was not detectable, and IGFBP-6 mRNA was low and inconsistent. In human fibroblasts, IGF-I treatment increased levels of IGFBP-3 and decreased levels of IGFBP-4 without influencing mRNA expression. IGF-I also increased steady state levels of IGFBP-5 mRNA. Dexamethasone alone decreased IGFBP-3, IGFBP-4, and IGFBP-5 mRNA, but it had no significant effect on IGFBP-3, -4, or -5 expression in the presence of IGF-I. Human fibroblast IGFBP-6 expression was stable under the different culture conditions; IGFBP-1 and -2 mRNA were not detected. These data demonstrate that IGF peptide and glucocorticoid individually modulate IGFBP expression and indicate that glucocorticoid has distinct effects on IGF regulation of IGFBP depending upon the particular IGFBP and the underlying mechanism of IGF regulation. Bovine fibroblasts may provide a useful model system to probe the molecular mechanisms of IGFBP-3, -4, and -5 gene expression and regulation by IGF-I and glucocorticoid, whereas human fibroblasts may be suitable for studying posttranscriptional interactions.