<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> (FGF<em>15</em>) has been proposed as a postprandial hormone that signals from intestine to liver to regulate bile acid and carbohydrate homeostasis. However, detecting FGF<em>15</em> in blood using conventional techniques has proven difficult. Here, we describe a stable isotope standards and capture by anti-peptide antibodies (SISCAPA) assay that combines immuno-enrichment with selected reaction monitoring (SRM) mass spectrometry to overcome this issue. Using this assay, we show that FGF<em>15</em> circulates in plasma in an FXR and circadian rhythm-dependent manner at concentrations that activate its receptor. Consistent with the proposed endocrine role for FGF<em>15</em> in liver, mice lacking hepatocyte expression of the obligate FGF<em>15</em> co-receptor, β-Klotho, have increased bile acid synthesis and reduced glycogen storage despite having supraphysiological plasma FGF<em>15</em> concentrations. Collectively, these data demonstrate that FGF<em>15</em> functions as a hormone and highlight the utility of SISCAPA-SRM as a sensitive assay for detecting low-abundance proteins in plasma.

This study tested the effect of fluid-induced shear on interleukin-6 expression in normal human articular chondrocytes in vitro. As determined by Northern blot analysis, interleukin-6 mRNA expression occurs in chondrocytes from osteoarthritic cartilage but not in normal chondrocytes. Applying fluid-induced shear stress to primary high density cultures of chondrocytes increased interleukin-6 mRNA signal 4-fold at 1 hour and 10 to <em>15</em>-fold at 48 hours compared with unsheared control cultures. At 48 hours, fluid-induced shear stress increased interleukin-6 protein levels in the culture medium 9 to 10-fold compared with unsheared controls. mRNA signals for interleukin-1alpha, interleukin-1beta, and tumor necrosis <em>factor</em>-alpha in RNA from sheared or control chondrocytes were not detected by Northern blotting. Transforming <em>growth</em> <em>factor</em>-beta mRNA signal was detectable but was not affected by shear. In contrast, human lung <em>fibroblasts</em> (WI-38) responded to fluid-induced shear with increased signal for transforming <em>growth</em> <em>factor</em>-beta, but not interleukin-6, mRNA. Both cell types did respond to interleukin-1alpha with increased interleukin-6 mRNA signal. These data demonstrated that distortional forces, such as fluid-induced shear stress, alter interleukin-6 levels in normal chondrocytes in vitro and suggest that increased interleukin-6 expression in osteoarthritic cartilage may result, in part, from alterations in the mechanical loading of the tissue.

c-Fos, a transcription <em>factor</em> that constitutes DNA-binding AP-1 complexes, regulates gene expression that promotes long-lasting cellular changes. We show that, in addition to its transcription <em>factor</em> activity, c-Fos regulates the metabolism of phospholipids cytoplasmically by an AP-1-independent activity. Two waves of c-Fos expression that promote subsequent waves of stimulation of 32P-orthophosphate incorporation into phospholipids are evidenced in quiescent cultured <em>fibroblasts</em> induced to re-enter the cell cycle. The first wave of c-Fos expression peaks at 7.5 min and returns to control levels by <em>15</em> min. The second wave starts by 30 min and remains elevated at 120 min. In the first wave, the lipids that incorporate 32P are predominantly second-messenger polyphosphoinositides (PIP, PIP2, PIP3); whereas in the second wave, membrane-biogenesis-related lipids (PI, PE, PA), become radioactive. Both waves of phospholipid activation depend on c-Fos expression. It is interesting that a peptide that blocks AP-1 nuclear import does not affect phospholipid activation. Immunocytochemical examination showed c-Fos immunoreactivity associated to the endoplasmic reticulum. We conclude that c-Fos, rapidly induced upon cell stimulation, associates to the endoplasmic reticulum where it first regulates the synthesis/ replenishment of phospholipids required for signal transduction pathways and subsequently regulates enzymes involved in the genesis of new membrane necessary for cell <em>growth</em>.

OBJECTIVE

Little is known about the effects of the vitamin D receptor (VDR) on hepatic activity of human cholesterol 7α-hydroxylase (CYP7A1) and cholesterol metabolism. We studied these processes in mice in vivo and mouse and human hepatocytes.

METHODS

Farnesoid X receptor (Fxr)(-/-), small heterodimer partner (Shp)(-/-), and C57BL/6 (wild-type control) mice were fed normal or Western diets for 3 weeks and were then given intraperitoneal injections of vehicle (corn oil) or 1α,25-dihydroxyvitamin D3 (1,25[OH]2D3; 4 doses, 2.5 μg/kg, every other day). Plasma and tissue samples were collected and levels of Vdr, Shp, Cyp7a1, Cyp24a1, and rodent <em>fibroblast</em> <em>growth</em> <em>factor</em> (Fgf) <em>15</em> expression, as well as levels of cholesterol, were measured. We studied the regulation of Shp by Vdr using reporter and mobility shift assays in transfected human embryonic kidney 293 cells, quantitative polymerase chain reaction with mouse tissues and mouse and human hepatocytes, and chromatin immunoprecipitation assays with mouse liver.

RESULTS

We first confirmed the presence of Vdr mRNA and protein expression in livers of mice. In mice fed normal diets and given injections of 1,25(OH)2D3, liver and plasma concentrations of 1,25(OH)2D3 increased and decreased in unison. Changes in hepatic Cyp7a1 messenger RNA (mRNA) correlated with those of Cyp24a1 (a Vdr target gene) and inversely with Shp mRNA, but not ileal Fgf<em>15</em> mRNA. Similarly, incubation with 1,25(OH)2D3 increased levels of Cyp24a1/CYP24A1 and Cyp7a1/CYP7A1 mRNA in mouse and human hepatocytes, and reduced levels of Shp mRNA in mouse hepatocytes. In Fxr(-/-) and wild-type mice with hypercholesterolemia, injection of 1,25(OH)2D3 consistently reduced levels of plasma and liver cholesterol and Shp mRNA, and increased hepatic Cyp7a1 mRNA and protein; these changes were not observed in Shp(-/-) mice given 1,25(OH)2D3 and fed Western diets. Truncation of the human small heterodimer partner (SHP) promoter and deletion analyses revealed VDR-dependent inhibition of SHP, and mobility shift assays showed direct binding of VDR to enhancer regions of SHP. In addition, chromatin immunoprecipitation analysis of livers from mice showed that injection of 1,25(OH)2D3 increased recruitment of Vdr and rodent retinoid X receptor to the Shp promoter.

CONCLUSIONS

Activation of the VDR represses hepatic SHP to increase levels of mouse and human CYP7A1 and reduce cholesterol.

The mechanisms of pulmonary repair in acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are poorly known. Hepatocyte <em>growth</em> <em>factor</em> (HGF) and keratinocyte <em>growth</em> <em>factor</em> (KGF) are key <em>factors</em> involved in alveolar epithelial repair, present in the bronchoalveolar lavage fluid (BALF) from patients with ALI/ARDS. The role of BALF mediators in their production remains to be determined. We evaluated the overall effect of BALF from 52 patients (27 ventilated patients with ALI/ARDS, 10 ventilated patients without ALI, and <em>15</em> nonventilated control patients) on HGF and KGF synthesis by lung <em>fibroblasts</em>. <em>Fibroblasts</em> were cultured in the presence of BALF. HGF and KGF protein secretion was measured using ELISA, and mRNA expression was evaluated using quantitative real-time RT-PCR. Only BALF from ALI/ARDS patients upregulated both HGF and KGF mRNA expression and protein synthesis (+271 and +146% for HGF and KGF, respectively). BALF-induced HGF synthesis from ALI/ARDS patients was higher than that from ventilated patients without ALI (P < 0.05). HGF secretion was correlated with BALF IL-1beta levels (rho = 0.62, P < 0.001) and BALF IL-1beta/IL-1 receptor antagonist ratio (rho = 0.54, P < 0.007) in the ALI/ARDS group. An anti-IL-1beta antibody partially (>50%) inhibited the BALF-induced HGF and PGE(2) secretion, whereas NS-398, a specific cyclooxygenase-2 (COX-2) inhibitor, completely inhibited it. Anti-IL-1beta antibodies as well as NS-398 reversed the COX-2 upregulation induced by BALF. Therefore, IL-1beta is a main BALF mediator involved in HGF secretion, which is mediated through a PGE(2)/COX-2-dependent mechanism. BALF mediators may participate in vivo in the production of HGF and KGF by lung <em>fibroblasts</em> during ALI/ARDS.

Expression of connective tissue <em>growth</em> <em>factor</em> (CTGF), a member of the CCN gene family, is known to be significantly induced by mechanical stress. We have therefore investigated whether other members of the CCN gene family, including Cyr61 and Nov, might reveal a similar stress-dependent regulation. <em>Fibroblasts</em> <em>growing</em> under stressed conditions within a three-dimensional collagen gel showed at least a <em>15</em> times higher level of Cyr61 mRNA than cells <em>growing</em> under relaxed conditions. Upon relaxation, the decline of the Cyr61 mRNA to a lower level occurred within 2 h, and was thus quicker than the response of CTGF. The regulation was fully reversible when stress was reapplied. Thus, Cyr61 represents another typical example of a stress-responsive gene. The level of the Nov mRNA was low in the stressed state, but increased in the relaxed state. This CCN gene therefore shows an inverted regulation relative to that of Cyr61 and CTGF. Inhibition of protein kinases by means of staurosporine suppressed the stress-induced expression of Cyr61 and CTGF. Elevated levels of cAMP induced by forskolin mimicked the effects of relaxation on the regulation of Cyr61, CTGF and Nov. Thus, adenylate cyclase as well as one or several protein kinases might be involved in the mechanoregulation of these CCN genes.

Preoligodendrocytes have been described in cultures and tissue prints of adult human white matter (Armstrong et al., 1992). To characterize further these precursors of human oligodendrocytes, we have investigated whether they express genes playing a critical role in oligodendrocyte development. In the intact human brain, platelet-derived <em>growth</em> <em>factor</em> receptor alpha (PDGF alpha R) and myelin transcription <em>factor</em> 1 (MyTI) transcripts are expressed in 1-2% of cells of the oligodendrocyte lineage (OL), and clusters of such cells can be found in the periventricular region. Myelin basic protein transcripts containing exon 2 information (exon 2+ MBP), which are characteristic of the premyelinating stage, are detected in <em>15</em>-20% of OL cells in vivo. When OL cells are separated from human white matter and allowed to regenerate in vitro, a much larger proportion of these cells express developmentally regulated genes, while exon 2- MBP and proteolipid protein (PLP) transcripts characteristic of mature OL cells appear transiently downregulated. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), even in the presence of PDGF, does not promote DNA synthesis in these cultured OL cells. Yet bFGF induces human oligodendrocytes to regenerate their processes rapidly in vitro and to express O4 antigens as well as exon 2+ MBP, MyTI, and PLP transcripts. While bFGF accelerates early regenerative processes, it also maintains high expression of exon 2+ MBP transcripts in OL cells for up to 2 weeks in vitro. In contrast, high levels of insulin in the absence of bFGF allow accumulation of exon 2- MBP and PLP transcripts in most OL cells at 2-3 weeks in vitro. We propose that the myelinated human brain harbors a small pool of precursors of oligodendrocytes and that <em>growth</em> <em>factor</em>-regulated phenotypic plasticity rather than mitogenic potential accounts for the regeneration of oligodendrocytes in the initial stages of demyelinating diseases such as multiple sclerosis.

BACKGROUND

Platelet preparations for tissue healing are usually preactivated before application to deliver concentrated growth factors. In this study, the authors investigated the differences between nonactivated and thrombin-activated platelets in wound healing.

METHODS

The healing effects (i.e., wound closure, myofibroblast formation, and angiogenesis) of nonactivated and thrombin-activated platelets were compared in experimental wounds in diabetic (db/db) animals. In vitro, fibroblast phenotype and function were tested in response to platelets and activated platelets. No treatment served as a negative control.

RESULTS

Wounds treated with platelets reached 90 percent closure after 15 days, faster than activated platelets (26 days), and with higher levels of myofibroblasts and angiogenesis. In vitro, platelets enhanced cell migration and induced two-fold higher myofibroblast differentiation and contraction compared with activated platelets.

CONCLUSIONS

Platelets stimulate wound healing more efficiently compared with activated platelets by enhancing fibroblast differentiation and contractile function. Similar levels of growth factors may induce different biological effects when delivered "on demand" rather than in an initial bolus.

Transforming <em>growth</em> <em>factor</em> (TGF)-beta1 is a pleiotropic cytokine with many functions, including those related to <em>growth</em> modulation, immunosuppression, and pro-inflammation, in a wide variety of cell types. In this study, we investigated the ability of TGF-beta1 to regulate RANTES production by activated rheumatoid synovial <em>fibroblasts</em>. <em>Fibroblast</em>-like synoviocytes (FLS) were cultured in the presence of TGF-beta1 and IL-1beta, IL-<em>15</em>, TNFalpha, or IL-17, and the secretion of RANTES into culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA). Expression of RANTES encoded mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR), and NF-kappaB binding activity for RANTES transcription was determined by electrophoretic mobility shift assay (EMSA). We found that the concentrations of RANTES in synovial fluid (SF) from rheumatoid arthritis (RA) patients were lower than in SF from osteoarthritis (OA) patients, whereas the concentrations of TGF-beta1 were higher in RA SF than in OA SF. TGF-beta1 dose-dependently inhibited TNFalpha-induced production of RANTES protein and mRNA from RA FLS. Addition of RA SF with high-level TGF-beta1 mimicked the effect of TGF-beta1 on TNFalpha-induced RANTES production, which was inhibited by treatment with anti-TGF-beta1 neutralizing antibody. TGF-beta1 blocked the degradation of cytosolic IkappaB-alpha and the translocation of activated NF-kappaB to the nucleus. EMSA showed that the inhibitory effect of TGF-beta1 was associated with decreased binding of NF-kappaB to the RANTES promoter. These results suggest that elevated TGF-beta1 in rheumatoid synovial tissue may suppress joint inflammation by inhibiting RANTES secretion from synovial <em>fibroblasts</em>, thus blocking the infiltration of immune cells. These findings may provide an explanation for the mechanism by which TGF-beta1 regulates immune function in RA.

We have developed a serum-free medium for clonal <em>growth</em> of normal human muscle satellite cells (HMSC). It consists of an optimized nutrient medium, MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal <em>growth</em> <em>factor</em> (EGF), insulin, dexamethasone, bovine serum albumin, and fetuin. <em>Fibroblast</em> <em>growth</em> <em>factor</em> was needed with dialyzed fetal bovine serum (dFBS) as the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final medium without significant loss. Clonal <em>growth</em> of HMSC in MCDB 120 plus SF is as good as with <em>15</em>% serum and 0.5% chicken embryo or bovine pituitary extract. However, <em>growth</em> is further improved by use of a doubly-supplemented (DS) medium containing both SF and 5% dFBS. Clonal <em>growth</em> of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer <em>growth</em> is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little differentiation, even when grown to high densities. However, they retain the capacity for extensive fusion and synthesis of increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed <em>growth</em> responses were always those of muscle cells.

OBJECTIVE

This study was undertaken to identify the intracellular signaling pathway involved in induction of macrophage migration inhibitory factor (MIF) in human rheumatoid arthritis (RA) synovial fibroblasts.

METHODS

Human RA synovial fibroblasts were treated with concanavalin A (ConA), various cytokines, and inhibitors of signal transduction molecules. The production of MIF by synovial fibroblasts was measured in culture supernatants by ELISA. The expression of MIF mRNA was determined using reverse transcriptase polymerase chain reaction (RT-PCR) and real-time PCR. Phosphorylation of p38 mitogen-activated protein (MAP) kinase in synovial fibroblasts was confirmed using Western blotting. The expression of MIF and p38 MAP kinase in RA synovium was determined using dual immunohistochemistry.

RESULTS

The production of MIF by RA synovial fibroblasts increased in a dose-dependent manner after ConA stimulation. MIF was also induced by interferon-γ, CD40 ligand, interleukin-15, interleukin-1β, tumor necrosis factor-α, and transforming growth factor-β. The production of MIF by RA synovial fibroblasts was significantly reduced after inhibition of p38 MAP kinase. The expression of MIF and p38 MAP kinase was upregulated in the RA synovium compared with the osteoarthritis synovium.

CONCLUSIONS

These results suggest that MIF production was induced through a p38 MAP-kinase-dependent pathway in RA synovial fibroblasts.

A cellular and molecular approach was used to gain new insight into the pathogenesis of interstitial fibrosis in chronic purine aminonucleoside nephrosis (PAN) nephrosis. Thirty experimental rats (PAN rats) were given <em>15</em> mg/100 g body wt of i.p. PAN at time 0, followed by 4.3 mg/100 g body wt i.p. on days 20, 27 and 34; 25 control rats received i.p. saline at the same time intervals. All rats had a right unilateral nephrectomy within the first four days. Groups of control and PAN rats were killed at 21, 37, 52, 72 and 91 days. Renal sections were studied by immunofluorescence to quantitate interstitial macrophages, T lymphocytes and <em>fibroblasts</em>, and to characterize the deposition of the extracellular matrix (ECM) proteins (collagens I, III and IV, fibronectin and laminin) and the tissue inhibitor of the metalloproteinases (TIMP). Steady state concentrations of mRNA from the whole kidney for these ECM proteins, the metalloproteinases, TIMP, and transforming <em>growth</em> <em>factor</em> beta (TGF-beta 1) were quantitated by Northern blot analysis. Significant increases in the number of interstitial macrophages and T lymphocytes were found in the PAN rat groups compared to that in controls. All ECM proteins examined were quantitatively increased in the tubulo-interstitium of PAN rats. The pattern of distribution of some ECM proteins was also modified in experimental animals. TIMP was increased in the interstitium of PAN rats; at later times, TIMP was most prominent in sclerotic regions of the glomeruli and in tubular protein droplets. Northern blot analysis revealed increased steady-state mRNA levels for components of each of the ECM proteins, no change for the metalloproteinases--stromelysin or collagenase--and a marked increase for TIMP and TGF-beta 1 in PAN animals. The results of this study suggest that the diffuse interstitial fibrosis found in chronic PAN nephrosis results from both increased production of ECM proteins and decreased matrix degradation.

BACKGROUND

Mast cells (MC) are involved in a wide spectrum of disorders characterized by neovascularization and fibroproliferation. We and others recently reported that human MC are a source of basic fibroblast growth factor (b FGF-2), a potent angiogenic and mitogenic polypeptide, in several disease conditions, such as chronic inflammation, hemangioma, and benign cutaneous mastocytosis. These findings suggest that FGF-2 may be an important mediator of cell proliferation and angiogenesis associated with MC. Since MC are heterogeneous across species, it is unknown whether FGF-2 expression is a feature common to all MC, or whether FGF-2 expression by MC can be regulated. We therefore examined FGF-2 expression by MC in mouse tissue and MC lines.

METHODS

Immunostaining, RT-PCR, ELISA, immunoblot and Northern blot analyses were employed to study four murine MC lines for FGF-2 expression and its regulation by transforming growth factor-beta (TGF-beta), stem cell factor (SCF), and tumor necrosis factor-alpha (TNF-alpha).

RESULTS

Mouse tissue MC and three of four murine MC lines (CFTL-12, CFTL-15, ABFTL-3) express FGF-2 as judged by immunostaining, ELISA, Western blot and Northern blot analyses, and reverse transcription-polymerase chain reaction. While TNF-alpha appeared to downregulate FGF-2 mRNA levels, treatment with SCF or TGF-beta resulted in an increase in the expression of FGF-2 at mRNA level which can be attenuated by TNF-alpha. However, the concurrent increase in FGF-2 protein was negligible, possibly due to immaturity of these cell lines.

CONCLUSIONS

Expression of FGF-2 may be a ubiquitous feature of MC in other species in addition to humans, and can be selectively regulated by SCF, TGF-beta and TNF-alpha.

Cholesterol homeostasis is regulated by the liver X receptor (LXR) at the transcriptional level, but it remains unknown whether LXR can affect expression levels of intrahepatic lipolysis related gene. Recent evidence has demonstrated that <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21) regulates hepatic lipolysis and fatty acid utilization. In the present study, we examined the role of LXR in FGF21 gene expression associated with regulation of cross-talk signals between cholesterol and triglyceride metabolism in the liver. An in vivo cholesterol feeding test revealed that intake of excess cholesterol increased cholesterol catabolism related gene expression as well as fatty-acid biosynthesis related gene expression. Moreover, the accumulated cholesterol suppressed FGF21 and hormone-sensitive lipase (HSL) gene expression. After <em>15</em>-day cholesterol feeding, hepatic triglyceride concentrations were negatively correlated with expression levels of the FGF21 and HSL genes in the liver. An LXR agonist (TO-901317) repressed the FGF21 gene expression in mouse primary hepatocytes and HepG2 cells. A promoter deletion study and electrophoretic mobility shift assay revealed that the human FGF21 promoter has at least one LXR response element located from -37 to -22 bp. In summary, LXR represses FGF21 gene expression at the transcription level and might suppress lipolysis and lipid utilization to protect the liver from excess accumulation of toxic cholesterol.

OBJECTIVE

The purpose of this study was to determine whether estrogen suppresses matrix metalloproteinase-2 and -9 proenzyme expression by fibroblasts that are derived from the supportive connective tissue of the pelvic floor.

METHODS

A primary fibroblast culture that was developed from a biopsy specimen of the arcus tendineus was treated with interleukin-1 beta (10-15 ng/mL), transforming growth factor-beta 1 (5-15 ng/mL), 17 beta-estradiol (10(-9)-10(-7) mol/L), and Imperial Chemical Industries (ICI) 182 780 (10(-8)-10(-6) mol/L). Cellular and extracellular protein were analyzed by Western blotting and substrate zymography, respectively, for the effect of each treatment on the amount of pro-matrix metalloproteinase-2 and -9 and the membrane type 1 matrix metalloproteinase protein.

RESULTS

Both cellular and extracellular pro-matrix metalloproteinase-2 protein were increased by transforming growth factor-beta1 (P =.01) and decreased by estradiol (P <.001) and ICI 182 780 (P =.02 and.002, respectively). Membrane type 1 matrix metalloproteinase was not affected by estradiol, ICI 182 780, interleukin-1 beta, or transforming growth factor-beta 1. Extracellular pro-matrix metalloproteinase-9 was increased by the cytokines interleukin-1 beta (P <.001) and transforming growth factor-beta1 (P <.001) and decreased by estradiol (P <.001) and ICI 182 780 (P <.001).

CONCLUSIONS

The proenzymes of the tissue-degrading matrix metalloproteinases -2 and -9 are decreased by 17-beta estradiol and ICI 182 780.

The basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) gene locus is transcribed into a number of mRNA transcripts including an antisense mRNA derived from the opposite DNA strand of the bFGF gene. Expression of this natural antisense RNA has been implicated in regulation of the bFGF sense mRNA expression and turnover. In the present study we examined the developmental pattern of expression of the bFGF antisense transcript in fetal and postnatal rat tissues. Northern hybridization with a strand-specific cRNA probe detected a 1.5-kb polyadenylated antisense RNA in all tissues examined except brain, in which two transcripts were detected as a doublet of approximately 1.3-1.5 kb in size. The level of antisense transcript expression was markedly tissue- and age-dependent. In the developing brain, both sense and antisense transcripts were detected by Northern hybridization, but the pattern of their expression was inversely related. The 6.0-kb bFGF sense transcript increased in an age-dependent manner from days 3-30 of postnatal development while the antisense transcript decreased to nearly undetectable levels over the same period. In embryonic (days <em>15</em>-19) liver, kidney, heart and intestine bFGF antisense RNA expression was low but increased dramatically at parturition, rising 5-10-fold over fetal levels by 10 days of age, then declined slowly to a new steady-state level in adult tissues. The level of antisense RNA in these tissues was much higher than that of bFGF sense mRNA, which was undetectable by Northern analysis. Sense and antisense trancripts were also detected in midgestational (11.5 days) embryos by RT-PCR. Antisense expression did not increase when embryos were explanted and cultured for 48 h (9.5-11.5 days). The apparent reciprocal relationship between the abundance of sense and antisense bFGF transcripts in developing brain supports the possibility of a regulatory role for the antisense transcript in this tissue. There was no evidence for a reciprocal relationship between sense and antisense expression in the other tissues examined, indicating that the relationship between sense and antisense RNA expression may be tissue-specific.

We determined whether cartilage could be engineered from mesenchymal progenitor cells (MPCs) normally found in amniotic fluid. Mesenchymal amniocytes were isolated from ovine amniotic fluid samples (n = 5) and had their identity confirmed by immunocytochemistry. Cells were expanded and then cultured as micromass pellets (n = 5) in a chondrogenic medium containing transforming <em>growth</em> <em>factor</em>-beta2 (TGF-beta2) and insulin <em>growth</em> <em>factor</em>-1 (IGF-1) for 6-12 weeks. Pellets derived from fetal dermal <em>fibroblasts</em> (n = 4) were cultured under identical conditions. Additionally, expanded mesenchymal amniocytes were seeded onto biodegradable polyglycolic acid scaffolds (n = 5) and maintained in the same chondrogenic medium within a rotating bioreactor for 10-<em>15</em> weeks. Engineered specimens were analyzed quantitatively and compared with native fetal hyaline cartilage samples (n = 5). Statistical analysis was by the unpaired Student's t-test (p < 0.05). The isolated cells stained positively for vimentin and cytokeratins-8 and -18, but negatively for CD31. Micromass pellets derived from mesenchymal amniocytes exhibited chondrogenic differentiation by both standard and matrix-specific staining. In contrast, these findings could not be replicated in dermal <em>fibroblast</em>-based pellets. The engineered constructs derived from mesenchymal amniocytes similarly displayed histological evidence of chondrogenic differentiation and maintained their original size and three-dimensional architecture. Quantitative assays of the engineered constructs revealed lower concentrations of collagen type II, but similar amounts of glycosaminoglycans, elastin, and DNA, when compared to native fetal hyaline cartilage. We conclude that mesenchymal amniocytes can be used for the engineering of cartilaginous tissue in vitro. Cartilage engineering from the amniotic fluid may become a practical approach for the surgical treatment of select congenital anomalies.

<em>Fibroblast</em> migration from the peri-wound collagenous stroma into the fibrin-laden wound is critical for granulation tissue formation and subsequent healing. Previously we found that <em>fibroblast</em> transmigration from a collagen matrix into a fibrin matrix required fibronectin (FN). Integrins alpha4beta1, alpha5beta1, and alphavbeta3 and dermatan sulfate CD44 were required for this invasive migration. Here we demonstrated that syndecan-4, a transmembrane heparan sulfate (HS) proteoglycan, known to bind FN, is also required for <em>fibroblast</em> invasive migration of a fibrin/FN gel. This conclusion was based on <em>fibroblast</em> migration using two independent means of disrupting syndecan-4: heparinase degradation of HS glycosaminoglycans or suppression of syndecan-4 core protein with antisense oligodeoxynucleotides. Isolated syndecan-4 from these <em>fibroblasts</em> bound Hep II recombinant constructs FN III12-V<em>15</em>)FN III12-<em>15</em>)FN III12-14 but did not bind the IIICS (V) domain. Furthermore, platelet-derived <em>growth</em> <em>factor</em> (PDGF), which is required to stimulate <em>fibroblast</em> migration, markedly increased cell levels of syndecan-4 core protein in a time and concentration-dependent fashion. PDGF also induced upregulation of syndecan-4 at transcriptional level as determined by RT-PCR. These results demonstrate that syndecan-4 is essential for <em>fibroblast</em> invasive migration into fibrin clot and that PDGF, the stimulus for migration, induces increased syndecan-4 core protein expression.

BACKGROUND

Bronchial fibroblasts contribute to airway remodelling, including airway wall fibrosis. Transforming growth factor (TGF)-β1 plays a major role in this process. We previously revealed the importance of the mevalonate cascade in the fibrotic response of human airway smooth muscle cells. We now investigate mevalonate cascade-associated signaling in TGFβ1-induced fibronectin expression by bronchial fibroblasts from non-asthmatic and asthmatic subjects.

METHODS

We used simvastatin (1-15 μM) to inhibit 3-hydroxy-3-methlyglutaryl-coenzyme A (HMG-CoA) reductase which converts HMG-CoA to mevalonate. Selective inhibitors of geranylgeranyl transferase-1 (GGT1; GGTI-286, 10 μM) and farnesyl transferase (FT; FTI-277, 10 μM) were used to determine whether GGT1 and FT contribute to TGFβ1-induced fibronectin expression. In addition, we studied the effects of co-incubation with simvastatin and mevalonate (1 mM), geranylgeranylpyrophosphate (30 μM) or farnesylpyrophosphate (30 μM).

RESULTS

Immunoblotting revealed concentration-dependent simvastatin inhibition of TGFβ1 (2.5 ng/ml, 48 h)-induced fibronectin. This was prevented by exogenous mevalonate, or isoprenoids (geranylgeranylpyrophosphate or farnesylpyrophosphate). The effects of simvastatin were mimicked by GGTI-286, but not FTI-277, suggesting fundamental involvement of GGT1 in TGFβ1-induced signaling. Asthmatic fibroblasts exhibited greater TGFβ1-induced fibronectin expression compared to non-asthmatic cells; this enhanced response was effectively reduced by simvastatin.

CONCLUSIONS

We conclude that TGFβ1-induced fibronectin expression in airway fibroblasts relies on activity of GGT1 and availability of isoprenoids. Our results suggest that targeting regulators of isoprenoid-dependent signaling holds promise for treating airway wall fibrosis.

Keratinocyte <em>growth</em> <em>factor</em>/<em>fibroblast</em> <em>growth</em> <em>factor</em>-7 (KGF/FGF-7) is an established paracrine mediator of hormone-regulated epithelial <em>growth</em> and differentiation. In all organs studied, KGF is uniquely expressed in cells of mesenchymal origin. To determine whether KGF and its receptor, keratinocyte <em>growth</em> <em>factor</em> receptor (KGFR) or <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor-2IIIb, were expressed in the porcine uterus as a potential paracrine system mediating progesterone action, we cloned KGF and KGFR partial cDNAs from the porcine endometrium. KGF and KGFR expression was detected in endometrium by Northern blot hybridization. Interestingly, in situ hybridization results demonstrated that KGF was expressed by endometrial epithelia and was particularly abundant between Days 12 and <em>15</em> of the estrous cycle and pregnancy. KGF secretion into the lumen of the porcine uterus was also detected on Day 12 of the estrous cycle and pregnancy. KGFR was expressed in both endometrial epithelia and conceptus trophectoderm. These novel findings suggest that KGF may act on the uterine endometrial epithelium in an autocrine manner and on the conceptus trophectoderm in a paracrine manner in the pig, which is the only species possessing a true epitheliochorial type of placentation.

Hepatocyte <em>growth</em> <em>factor</em> (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 <em>factor</em>, 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 <em>factor</em> 1, and upstream stimulatory <em>factor</em>, and together, they constitute a multifunctional composite binding site through which these different transcription <em>factors</em> exert their regulatory effects on HGF promoter activity. Functional assays revealed that PPARgamma, with its ligand, <em>15</em>-deoxy-prostaglandin J2, strongly stimulates HGF promoter activity. On the other hand, nuclear <em>factor</em> 1, activating protein-2, and chicken ovalbumin upstream promoter-transcription <em>factor</em> transcription <em>factors</em> 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, <em>15</em>-deoxy-prostaglandin J2, induces endogenous HGF mRNA and protein expression in <em>fibroblasts</em> in culture.

Tumor necrosis <em>factor</em> (TNF) activates both p42 and p44 mitogen-activated protein kinases (MAPK) in human FS-4 <em>fibroblasts</em>, cells for which TNF is mitogenic. We now show that TNF activates p42 MAPK in two cell lines whose <em>growth</em> is inhibited by TNF. A mutant TNF that binds only to the p55 TNF receptor (TNFR) produced a similar degree of activation as wild-type TNF in FS-4 <em>fibroblasts</em>, indicating that the p55 TNFR is sufficient to mediate p42/p44 MAPK activation. The upstream intracellular signals that couple the TNFR to MAPK activation are still poorly defined. We now show that neither phorbol ester-sensitive protein kinase C nor Gialpha link TNF to p42/p44 MAPK activation, because pretreatment of FS-4 cells with phorbol ester to down-regulate protein kinase C or pretreatment with pertussis toxin to block Gialpha does not inhibit p42/p44 MAPK activation by TNF. To further analyze MAPK activation in FS-4 cells, we compared p42/p44 MAPK activation by TNF and epidermal <em>growth</em> <em>factor</em> (EGF). While tyrosine phosphorylation of p42/p44 MAPK was detected almost immediately (30 s) after stimulating cells with EGF, TNF-induced tyrosine phosphorylation was detected only after a more prolonged time interval (initially detected at 5 min and peaking at <em>15</em>-30 min). In addition, the anti-inflammatory drug sodium salicylate, previously demonstrated to inhibit NF- kappaB activation by TNF, blocked the activation of p42/p44 MAPK in response to TNF but not in response to EGF. These findings demonstrate that the TNF and EGF receptors utilize distinct signaling molecules to couple to MAPK activation. Elucidation of the mechanism whereby sodium salicylate blocks TNF-induced p42/p44 MAPK activation may help to clarify TNF-activated signaling pathways.

Epidermal <em>Growth</em> <em>Factor</em> (EGF) and Tetradecanoyl Phorbol Acetate (TPA) initiate signalling cascades in C3H 10T1/2 <em>fibroblasts</em> by primarily activating distinct protein kinases, the EGF receptor tyrosine kinase and protein kinase C respectively; there is no signal crossover at the initiation of signalling. Nevertheless, we show here that both agents rapidly elicit common intracellular responses, including the phosphorylation of complexed and chromatin-associated forms of a 33 kDa phosphoprotein (pp33), that of a <em>15</em> kDa chromatin-associated phosphoprotein (pp<em>15</em>), as well as the transcriptional activation of a common subset of genes including the c-fos proto-oncogene. 2-aminopurine specifically abolishes complexed and chromatin-associated pp33 phosphorylation in response to EGF and TPA, as well as the induction of c-fos by both agents. The activation of protein kinase C and the levels of transcription <em>factors</em> that bind to the serum response element (SRE), TPA response element (TRE) or NFkB sites in stimulated cells are relatively unaffected by 2-aminopurine. This, to our knowledge, is the first demonstration that it is possible, by using 2-aminopurine which selectively blocks TPA-stimulated pp33 phosphorylation, to block c-fos induction in TPA-treated cells although protein kinase C remains fully active. Further, we show here that although EGF- and TPA-stimulated induction of c-fos is abolished by 2-aminopurine, the appearance of TRE-binding activity in nuclear extracts of stimulated cells is unaffected, suggesting that EGF- and TPA-stimulated induction of TRE-binding activity utilises existing proteins and is not dependent on fresh c-FOS synthesis. These results imply that 2-aminopurine-sensitive complexed and chromatin-associated pp33 phosphorylation may be crucial to c-fos induction in response to EGF and TPA.

CD123 (alpha-subunit of IL-3 receptor) expression on primitive and committed human hematopoietic cells was studied by multicolor sorting and single-cell culture. The sources of cells included fetal liver (FLV), fetal bone marrow, umbilical cord blood, adult bone marrow and mobilized peripheral blood. Three subsets of CD34+ cells were defined by the levels of surface CD123: CD123negative, CD123low, and CD123bright. Coexpression of lineage markers showed that a majority of CD34+CD123bright cells were myeloid and B-lymphoid progenitors, while erythroid progenitors were mainly in the CD34+CD123negative subset. The CD34+CD123low subset contained a heterogeneous distribution of early and committed progenitor cells. Single CD34+ cells from the CD123 subsets were cultured in a cytokine cocktail of stem cell <em>factor</em>, interleukin 3 (IL-3), IL-6, GM-CSF, erythropoietin, insulin-like <em>growth</em> <em>factor</em>-1, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. After 14 days of incubation, a higher cloning efficiency (CE) was observed in the CD34+CD123negative and CD34+CD123low fractions (37+/-23% and 44+/-23%, respectively) than in the CD34+CD123bright fraction (<em>15</em>+/-21%). Using previously published criteria that colonies containing dispersed, translucent cells (dispersed <em>growth</em> pattern, DGP) were derived from primitive cells and that colonies composed solely of clusters were from committed cells, early precursors were distributed evenly in the CD34+CD123negative and CD34+CD123low subsets. When CD38 and CD90 (Thy-1) were used for further characterization of CD34+ cells from FLV, CE increased from 37+/-23% in CD123negative to 70+/-19% in CD123negativeCD38- and from 44+/-23% in CD123low to 66+/-19% in CD123lowCD38-. No significant increase in CE or DGP progenitors was observed when CD34+ cells were sorted by CD90 and CD123. We concluded that: A) high levels of CD123 were expressed on B-lymphoid and myeloid progenitors; B) early erythroid progenitors had little or no surface CD123, and C) primitive hematopoietic cells are characterized by CD123negative/low expression.