Generation of contractile forces as <em>fibroblasts</em> attach and migrate through collagenous substrates is a fundamental behavior, yet its regulation and consequences are obscure. Although the transforming <em>growth</em> <em>factor</em>-betas (TGF-beta) are similarly important in fibrosis and tissue repair, their role in contraction is controversial. Using a quantitative, 3D collagen culture model we have measured the effects of TGF-beta1 and -beta3 on contractile forces generated by human dermal <em>fibroblasts</em>. Maximal stimulation was between 7.5 and <em>15</em> ng/ml of TGF-beta1. Higher doses were inhibitory (30 ng/ml), giving a bell-shaped dose response. The initial rate of force generation was increased sevenfold (<em>15</em> ng/ml). A similar response pattern was seen with TGF-beta3 alone. However, the addition of both isoforms together stimulated a biphasic increase in force generation, suggesting that there was a distinct temporal cooperativity between the two isforms. This very early onset (10-20 min) of stimulation suggested that TGF-beta might act through cell attachment and integrin function and the effect of TFG-beta on expression of fibronectin (FnR) and vitronectin (VnR) integrin receptors was monitored over the same time scale. TGF-beta1 dramatically up-regulated VnR expression, relative to FnR, over time but the optimal time for this was 2-4 h later than that of force stimulation. It is concluded that TGF-beta1 and -beta3 behave here primarily as mechanoregulatory <em>growth</em> <em>factors</em> and that stimulation of integrin expression may be a consequence of the altered cell stress.

Retinal pigment epithelium transplantation has been proposed as adjunctive treatment for age-related macular degeneration following surgical excision of choroidal neovascular membranes. The goal of this study was to develop a model to evaluate retinal pigment epithelium transplantation onto human Bruch's membrane in vitro. We investigated the ability of cultured fetal human retinal pigment epithelium to colonize human cadaver Bruch's membrane, determined the incubation time needed to form a monolayer and to exhibit apical microvilli and tight junctions, and assessed the production of basement membrane. Freshly enucleated (less than 48 hours old) human eyes were cut through the pars plana, and the anterior segment, vitreous, and retina were removed. The native retinal pigment epithelium was debrided with a surgical sponge. Bruch's membrane and choroid at the macula were trephined with a 7.0 mm diameter trephine and then incubated with 1/2 ml of Dulbecco's modified Eagle's medium +<em>15</em>% fetal calf serum+basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (1 ng ml-1), and fetal human retinal pigment epithelium at a concentration of 242,000 cells ml-1. Specimens were incubated for 1, 4, 6, 8, 12, or 24 hours. The specimens were fixed in half strength Karnovsky's fixative, processed, and analysed with scanning and transmission electron microscopy. The retinal pigment epithelium covered the debrided macular specimens to different degrees at different incubation times. After 1 hour, the cells started to attach and flatten (median percent coverage: 78%). The extent of Bruch's membrane coverage by fetal retinal pigment epithelium varied greatly between specimens. After 4-6 hours, the cells covered the entire debrided surface in a monolayer (median percent coverage: 97.2% at 4 hours, 99.8% at 6 hours). Tight junctions were observed, and the cells had few apical microvilli. The lateral cell borders were obliquely oriented with respect to Bruch's membrane, and the nuclei were elongated, exhibited prominent nucleoli, and were oriented parallel to Bruch's membrane. After 6-8 hours, cells started to become hexagonal (median percent coverage at 8 hours: 99.97%). Cells attached to the inner collagenous layer tended to be flatter than cells attached to residual native basement membrane. At 12 and 24 hours, expression of hexagonal shape, tight junctions, and apical microvilli were observed more frequently (median percent coverage: 99.87% at 12 and 100% at 24 hours). No newly formed basement membrane was observed at these time points. In separate experiments comparing attachment in the presence and absence of native RPE basement membrane, the presence of native retinal pigment epithelial basement membrane promoted the early attachment of the cells and more rapid expression of normal morphology. This in vitro system provides a reproducible way to study the adherence of retinal pigment epithelium to normal and diseased human Bruch's membrane.

The aim of the present study was to assess the frequency and <em>factors</em> associated with the progression of vascular calcifications (VCs) using a semiquantitative X-ray score. We included all prevalent hemodialysis patients with initial radiological scores ranging from 0 to 3 according to the severity of the VCs. Patients were classified as non-progressors or progressors after 3 years. Among the 85 patients, 44.7% were classified as progressors. Only exhibiting high levels of serum intact parathyroid hormone (PTH, >190 pg/ml) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-23 levels (>3,000 RU/ml) is associated with the risk of VC progression (OR 5.8, 95% CI 1.7-19.8, p = 0.004). Calcitriol analogs (38%), cinacalcet (<em>15</em>%), dialysate calcium (mean 1.48 mmol/l), dialysis session time (4-8 h) and calcium- (10%) and non-calcium-based phosphate binders (38%) were prescribed on an individual basis. Hyperphosphatemia (<10%) and, especially, hypercalcemia (1%) and hyperparathyroidism (>585 pg/ml = 0%) were infrequently observed. In conclusion, the main <em>factor</em> associated with VC progression was the association of higher serum PTH and FGF-23 levels. It remains to be seen whether patients should be treated to lower their PTH value, even within the target range, using calcitriol analogs, calcimimetics, parathyroidectomy, or by modifying the Klotho-FGF-23 axis.

OBJECTIVE

Renal interstitial fibrosis is a hallmark of progressive chronic kidney disease (CKD). Previous studies reported that kruppel-like <em>factor</em> <em>15</em> (KLF<em>15</em>) is an important regulator of cardiac fibrosis and could reduce the expression of extracellular matrix in mesangial cells. However, the role of this transcription <em>factor</em> in renal interstitial fibrosis has not been reported.

METHODS

In this study, we examined KLF<em>15</em> expression in the remnant kidney of 5/6 nephrectomized rats 12 or 24 weeks after operation. In vitro we examined the effect of altered KLF<em>15</em> expression on the production of extracellular matrix and the pro-fibrotic <em>factor</em> CTGF in rat renal fibroblasts (NRK-49F), and further explored the related mechanisms.

RESULTS

The level of KLF<em>15</em> was drastically decreased in the renal interstitium of 5/6 nephrectomized rats with progressive interstitial fibrosis, especially at 24 weeks. Our in vitro evidence showed that overexpression of KLF<em>15</em> repressed basal and transforming growth <em>factor</em>-β1 (TGF-β1)-induced extracellular matrix and CTGF in NRK-49F cells. In addition, TGF-β1-mediated activation of extracellular-regulated kinase (ERK) / mitogen-activated protein kinase (MAPK) and Jun N-terminal kinase (JNK) /MAPK downregulated KLF<em>15</em> expression and increased the level of extracellular matrix and CTGF, and all these effects were completely abolished by ERK1/2 inhibitor and JNK inhibitor in NRK-49F cells.

CONCLUSIONS

Our findings implicate that KLF<em>15</em> plays an important role and may prove to be an antifibrotic <em>factor</em> in renal interstitial fibrosis through regulation of ERK/MAPK and JNK/MAPK signaling pathways.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists are increasingly used in patients with diabetes, and some studies have suggested a beneficial effect on organ fibrosis, but their effects on dermal cell <em>growth</em> and extracellular matrix (ECM) turnover are unknown. To investigate the effect of the PPAR-gamma agonist troglitazone on cell <em>growth</em> and matrix production in human dermal <em>fibroblasts</em> (HDF), HDF were cultured and grown in a different concentration of troglitazone. PPAR-gamma expression and matrix production were measured in HDF in the presence of troglitazone. The mRNA expressions of TGF-beta1, collagen I (Col I) and fibronectin (FN) were determined by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). The protein of transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1) was determined by enzyme-linked immunosorbent assay (ELISA) and proteins of Col I and FN were determined by Western blotting. The mRNA expression of TGF-beta1, Col I and FN were significantly decreased in HDF in <em>15</em>-30 micromol l(-1) troglitazone compared to the control group with Dulbecco's modified Eagle's medium (P<0.01). An obvious decrease of TGF-beta1 protein was found in troglitazone-treated groups as compared to the control group (P<0.05). Exposure of HDF to troglitazone reduced col I secretion (P<0.05), and fibronectin secretion (P<0.05). This study suggests that PPAR-gamma agonist will provide a novel approach with therapeutic potential in dermal fibrosis, such as hypertrophic scar, keloid and so on.

The biosynthesis of the receptor for platelet-derived <em>growth</em> <em>factor</em> (PDGF) was examined in metabolically labeled human foreskin <em>fibroblasts</em>. The receptor was synthesized as a 145-kDa precursor, which, when incubated with endo-beta-N-acetylglucosaminidase H (endo H), underwent a <em>15</em>-kDa decrease in molecular mass. This indicates that the size of the core protein is about 130 kDa and that the 145-kDa form represents a receptor precursor carrying high-mannose N-linked oligosaccharide groups. Within <em>15</em> min after synthesis, the receptor was converted to a 165-kDa form. This form was entirely resistant to endo H treatment and probably represents a receptor molecule that has undergone further posttranslational modification, including O-linked glycosylation. Subsequently, within 30 min, a molecule of 170 kDa--i.e., the size of the mature receptor--appeared. A slightly larger molecule, of 175 kDa, which could be immunoprecipitated from PDGF-stimulated 32P-labeled cells, probably represents a receptor further modified by autophosphorylation. The 170-kDa molecule had an isoelectric point of about 4.5. Addition of PDGF increased the turnover rate of the 170-kDa PDGF receptor.

The normal contraction of open wounds and many forms of pathologic contracture are related by the presence of a contractile <em>fibroblast</em> known as a myo<em>fibroblast</em>. The function of this cell has been postulated as a result of previous pharmacological, immunological, and biochemical testing on strips of contracted connective tissue. The purpose of this study was to develop a specific assay that could measure the concentration of one contractile element (actin) within cultured myo<em>fibroblasts</em> isolated from a contracting wound and in normal <em>fibroblasts</em> from uninjured dermis. Rates of <em>growth</em> and actin concentration through <em>15</em> days of culture were compared among populations of paired control <em>fibroblasts</em> from normal dermis and granulating wound myo<em>fibroblasts</em> from three patients. <em>Growth</em> curves showed that myo<em>fibroblasts</em> always grew slower than <em>fibroblasts</em>. An enzyme-linked immunosorbent assay showed that actin concentration was generally greater in mass cultures of granulating wound myo<em>fibroblasts</em> than in <em>fibroblasts</em> from uninjured dermis. During exponential <em>growth</em> (1-6 days) the average actin concentration among myo<em>fibroblast</em> lines ranged from 24 to 62 pg/cell. Average actin levels among control <em>fibroblasts</em> ranged from 3 to 47 pg/cell during the same interval. After <em>15</em> days of culture, actin concentration peaked twice. The first actin peak occurred within the period of exponential <em>growth</em>. At confluency, cellular actin levels dropped. Superconfluent cultures exhibited a second actin peak that displayed an irregular pattern of actin concentration. The latter observation suggested an artifact that might be the result of three-dimensional matrix of cells that altered points of cell adhesion and produced an irregular pattern of actin concentration. These data show that the phenotype of increased actin in cultured myo<em>fibroblasts</em> was carried over by myo<em>fibroblasts</em> from contracted skin wounds to culture. Because of a higher concentration of actin in myo<em>fibroblasts</em> than in undifferentiated <em>fibroblasts</em>, these data suggest that the differentiation process of myo<em>fibroblasts</em> may be associated with an increased availability of monomeric actin for filament synthesis. This study demonstrates that the use of tissue culture and our enzyme-linked immunosorbent assay will be a useful method to study <em>factors</em> affecting myo<em>fibroblast</em> phenotypic modulation. Future studies should be directed toward developing procedures for isolation of pure populations of myo<em>fibroblasts</em> as well as extracellular matrices that would maintain the morphology of both differentiated myo<em>fibroblasts</em> and normal undifferentiated <em>fibroblasts</em>.

The expression of angiogenic <em>factors</em> may represent useful markers for diagnosis and prediction of disease outcome. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (b-FGF) is a potent angiogenic <em>factor</em> which promotes in vitro <em>growth</em> of endothelial cells and in vivo vessel formation. We investigated the expression of b-FGF in patients affected with malignant and non-malignant pleural diseases and presenting clinically with non-specific signs and symptoms. We also studied the relationships between the expression of b-FGF in patients with malignant pleural mesothelioma (MM) and tumour aggressiveness, assessed as tumour vessel density (TVD), or patient survival. Basic-FGF was measured by immunoassay in the serum and pleural effusions (PE) of 37 patients. Of these, MM was diagnosed in <em>15</em>/37 patients while the remaining patients had either peripheral lung adenocarcinoma (PLA) or benign inflammatory pleural disease (BPD). The mean b-FGF level measured 8.5+/-6.1 pg/ml in the PE of the malignant group (MM + PLA) and 23.9+/-19.8 in the PE of the non-malignant group (BPD) (p=0.001). The mean b-FGF level was significantly lower in the PE of MM patients (6.9+/-5.2 pg/ml) compared to BPD patients (p=0.004). Linear regression analysis showed a significant inverse correlation (r=-0.59; p=0.041) between b-FGF levels found in MM PE and patient survival. A noteworthy relationship between high serum b-FGF levels and reduced survival was also observed (r=-0.57; p=0.052). Interestingly, both serum (r=0.48; p=0.114) and PE (r=0.26; p=0.413) b-FGF levels in MM patients correlated poorly with TVD. Our data indicate that b-FGF is significantly more expressed in non-malignant compared to malignant PE, this difference being particularly evident between MM and BPD. Our results also suggest that high b-FGF levels correlate with poor MM patient survival through mechanisms which may be independent of b-FGF angiogenic potential.

Using quantitative in situ hybridization and immunohistochemistry the expression of acidic and basic <em>fibroblast</em> <em>growth</em> <em>factors</em> (aFGF, bFGF) in dorsal root ganglia (DRGs) was examined. Around 5% of the small neurons expressed bFGF mRNA in normal DRGs. Nerve injury induced a very dramatic and rapid up-regulation in bFGF mRNA levels, and around 80% of all DRG neurons expressed bFGF mRNA 3 days after axotomy. A distinct increase in bFGF-like immunoreactivity (LI) was also detected as early as <em>15</em> h after axotomy. The elevation of bFGF mRNA and protein levels declined after 1 week. bFGF mRNA was also up-regulated in non-neuronal cells following axotomy. Normally bFGF-LI was mainly localized in the nuclei of DRG neurons and in some non-neuronal cells. After nerve section, bFGF-LI was in addition found in the cytoplasm, and many more bFGF-positive non-neuronal cells were observed. By means of confocal microscopy analysis of axotomized DRGs, some bFGF-LI could be detected in vesicle-like structures in the cytoplasm as well as in the nucleoli, in addition to the nuclear location. Application of leukaemia inhibitory <em>factor</em> to the transected sciatic nerve significantly increased the number of bFGF-positive neurons, whereas the bFGF-LI in non-neuronal cells was strongly suppressed. About 70% of the normal DRG neurons expressed aFGF mRNA and aFGF-LI. Axotomy produced a moderate increase in aFGF mRNA levels, but no detectable effect on protein levels. Taken together, the results show that bFGF may be involved in the neuronal response to injury and suggest a role in neuronal survival and regeneration in axotomized DRG neurons.

The effect of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) on the <em>growth</em> of chondrocytes in soft agar was examined. FGF induced colony formation by chick embryo and rabbit chondrocytes. The colony-forming efficiency of FGF-exposed chondrocytes was similar to that of Rous sarcoma virus-transformed chondrocytes (<em>15</em>-20%). Other mitogenic agents tested, such as epidermal <em>growth</em> <em>factor</em>, insulin, insulin-like <em>growth</em> <em>factor</em>-l, and platelet-derived <em>growth</em> <em>factor</em>, induced very low levels of colony formation. The induction of <em>growth</em> in soft agar of chondrocytes by FGF was not due to cells' phenotypic transformation, because chondrocytes grown in soft agar with FGF retained the ability to synthesize cartilage-characteristic proteoglycan. FGF did not induce <em>growth</em> in soft agar of chondrocytes whose phenotypic expression was suppressed by retinoic acid or 5-bromodeoxyuridine. In addition, FGF did not induce <em>growth</em> in soft agar of primary <em>fibroblasts</em> and normal rat kidney (NRK) cells. These results suggest that FGF selectively stimulates <em>growth</em> of differentiated chondrocytes in soft agar.

BACKGROUND

Fibroblast growth factor (FGF-23) is a novel phosphaturic factor. Current data suggest that serum phosphate, dietary phosphate and 1,25 dihydroxyvitamin D regulate circulating FGF-23 levels in vivo. We examined if hypogonadism-induced increases in serum phosphate are associated with increases in circulating FGF-23 in healthy men in the absence of dietary manipulation.

METHODS

25 healthy men were administered goserelin acetate (GnRH analog) 3.6 mg subcutaneously every 4 weeks for 12 weeks to induce acute testosterone and estrogen deficiency. Subjects consumed an ad libitum diet. Morning fasting blood and urine samples were collected to measure serum phosphate, serum intact FGF-23, PTH, and the maximum tubular reabsorption of phosphate (T(m)P/GFR) at baseline, weeks 4 and 12. The changes in serum FGF-23 and phosphate at weeks 4 and 12 were compared to baseline using paired t-tests.

RESULTS

Goserelin therapy decreased mean serum testosterone levels from 543+/-160 ng/dL to 33+/-15 ng/dL at week 4 (p<0.001), and to 20+/-10 ng/dL at week 12 (p<0.001). Serum phosphate increased significantly from 3.4+/-0.6 mg/dL to 3.9+/-0.4 mg/dL at week 4 (p=0.002), and to 4.3+/-0.4 mg/dL at week 12 (p<0.001). T(m)P/GFR increased significantly from 3.2+/-0.6 mg/dL to 3.6+/-0.5 mg/dL at week 4 (p<0.004), and to 4.1+/-0.6 mg/dL at week 12 (p<0.001). FGF-23 levels, however, did not change during the 12-week study.

CONCLUSIONS

Gonadal steroid deprivation increased serum phosphate levels in men but did not affect serum FGF-23 concentrations. The absence of any change in circulating FGF-23 suggests that supraphysiologic levels of serum phosphate may be required to stimulate circulating FGF-23 or that FGF-23 production is primarily sensitive to changes in dietary phosphate or 1,25 dihydroxyvitamin D within this physiologic serum phosphate range.

Autoradiographic methods were used to show that non-neuronal cells from dissociated chick ciliary ganglia grown in cell culture for 1 day exhibit high affinity uptake for norepinephrine (NE) and/or specific receptors for nerve <em>growth</em> <em>factor</em> (NGF). Using immunofluorescence procedures, it was demonstrated that these cells reacted neither with the neuron-specific marker tetanus toxin nor with antibodies to the <em>fibroblast</em> marker fibronectin. The cells were, however, positive for 04 antigen, which is present on Schwann cells and oligodendrocytes and is recognized by a monoclonal antibody (Schachner, M., S. K. KIm, and R. Zehnle (1981) Dev. Biol. 83: 328-338). At all stages studied between embryonic day 6 (E6) and embryonic day 14 (E14), about 80% of the non-neuronal cells were positive for 04 antigen, the other non-neuronal cells being identified as <em>fibroblasts</em> or <em>fibroblast</em>-like cells by staining with antibodies to fibronectin. The proportion of cells with NGF receptors and cells with NE uptake decreased during development between E6 and E14. The percentage of 04-positive cells which have NGF receptors decreased from about 95% at E6 to about 35% at E14. The proportion of 04-positive cells with NE uptake decreased from about 57% at E6 to about <em>15</em>% at E14. Thus, a considerable proportion of the non-neuronal cell population in embryonic ciliary ganglia displays neuronal properties. We suggest that those cells exhibiting biochemical properties of both differentiated glial cells and neurons are precursor cells which have the potential to develop either into glial cells or neurons.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) mutations have been found in craniosynostosis syndromes with and without limb and/or dermatologic anomalies. Ocular manifestations of FGFR2 syndromes are reported to include shallow orbits, proptosis, strabismus, and hypertelorism, but no ocular anterior chamber, structural abnormalities have been reported until now. We evaluated three unrelated patients with severe Crouzon or Pfeiffer syndrome. Two of them had ocular findings consistent with Peters anomaly, and the third patient had opaque corneae, thickened irides and ciliary bodies, and shallow anterior chambers with occluded angles. Craniosynostosis with and without cloverleaf skull deformity, large anterior fontanelle, hydrocephalus, proptosis, depressed nasal bridge, choanal stenosis/ atresia, midface hypoplasia, and elbow contractures were also present. These patients had airway compromise, seizures, and two died by age <em>15</em> months. All three cases were found to have the same FGFR2 Ser351Cys (1231C to G) mutation predicted to form an aberrant disulfide bond(s) and affect ligand binding. Seven patients with isolated Peters anomaly, two patients with Peters plus syndrome, and three cases with typical Antley-Bixler syndrome were screened for this mutation, but none was found. These phenotype/genotype data demonstrate that FGFR2 is involved in the development of the anterior chamber of the eye and that the Ser351Cys mutation is associated with a severe phenotype and clinical course.

alpha-Thrombin alone is able to stimulate DNA synthesis reinitiation of G0-arrested Chinese hamster lung <em>fibroblasts</em> (CC139) as well as continued <em>growth</em> of these cells in serum-free medium. Although insulin at high concentrations (1-10 micrograms/ml) is not intrinsically mitogenic for these cells, it potently enhances the <em>growth</em>-promoting action of thrombin. The generation time of CC139 cells in the defined medium, transferrin, alpha-thrombin, insulin, is around <em>15</em> h. To determine whether this effect of insulin is mediated via putative receptors for the insulin-like <em>growth</em> <em>factors</em> (IGFs) on these cells, we examined the abilities of two IGFs, Multiplication-Stimulating Activity (MSA) and IGF-I, to potentiate the thrombin-induced reinitiation of DNA synthesis. Both IGFs were found to be as effective as insulin for this biological effect; however, much lower concentrations were required to elicit half-maximal response, 100 ng/ml of MSA and 30 ng/ml of IGF-I. Detailed binding studies using 125I-labelled insulin, MSA, and IGF-I revealed that CC139 cells specifically bind all three polypeptides with IC50 values for the corresponding ligands of 1-2 ng/ml, 80-100 ng/ml, and 30-40 ng/ml, respectively. 125I-MSA binding was insulin-insensitive, whereas insulin did compete with 125I-IGF-I for binding to CC139 cells. These results indicate that CC139 cells possess at least two types of IGF receptors, an insulin-insensitive IGF receptor with high affinity for MSA which apparently mediates its biological effect, and an insulin-sensitive IGF-I receptor. Insulin appears to exert its mitogen-potentiating activity in CC139 <em>fibroblasts</em> by interacting with the IGF-I receptor.

BACKGROUND

Pulmonary pleomorphic carcinoma (PPC) is a rare type of lung cancer characterized by the poor response to conventional chemotherapy and subsequent disappointing outcomes. Therefore, it is paramount to delineate the molecular characteristics of this disease entity.

METHODS

In this study, we retrospectively examined the surgical specimens of 61 patients who underwent lung surgery. Mutational or gene amplification statuses of epidermal growth factor receptor (EGFR), k-ras, c-kit, c-met, and fibroblast growth factor receptor (FGFR) were examined using genomic DNA sequencing, real-time PCR and/or fluorescence in situ hybridization (FISH).

RESULTS

The median age was 61 years, and 50 patients were men and 11 were women. In the histologic review of epithelial component, adenocarcinoma were in 44 cases (72%), squamous cell carcinoma in 15 (25%) and large cell carcinoma in 2 patients (3%). Overall, 30 cases (49%) had any molecular alterations. Nine patients (15%) possessed EGFR deletion in exon 19 (n = 8) or L858R mutations in exon 21 (n = 1), while 3 other cases having atypical EGFR mutations. Six patients (9.8%) had k-ras mutations in exon 12, and 3 had c-kit mutations. High gene copy number of c-met was found in 11 patients (18.0%) and that of FGFR was in 6 patients (9.8%). No significant relationships were identified among the occurrence and type of mutations and patient survival or any other clinicopathological variables.

CONCLUSIONS

Given the diverse repertoire of mutational profiles observed in PPC samples, clinical trials based on accurate cancer-genotyping should be considered as a legitimate treatment scheme for this rare disease entity in the future.

Three alternatively spliced forms of the amyloid precursor protein (APP), APP-695, APP-751, and APP-770, were expressed in the baculovirus expression vector system. The recombinant proteins were secreted into the culture medium by infected insect cells, and APP molecules were detected in insect cells and medium 2 days after infection with the recombinant APP-baculoviruses. A partial sequence of the NH2 terminus of the secreted protein revealed identity with the native secreted protein and showed that the signal peptide was recognized and properly cleaved in insect cells. Purified secreted recombinant APP-751 comigrated with protease nexin 2 purified from platelets and <em>fibroblasts</em>. A <em>15</em>-kDa COOH-terminal fragment of APP was also detected in cells infected with recombinant baculoviruses, suggesting that recombinant APP proteins were cleaved at the COOH-terminal end like native APP protein. Recombinant APP-751 and APP-770 formed complexes with epidermal <em>growth</em> <em>factor</em>-binding protein, whereas APP-695 did not. In addition, recombinant APP-751 and APP-770 inhibited trypsin and chymotrypsin activity, whereas APP-695 did not. <em>Growth</em> of a human <em>fibroblast</em> cell line, A-1, that required APP for complete <em>growth</em>, was restored upon addition of secreted recombinant APP-695 or APP-751. Thus, the appropriately sized, secreted recombinant APP proteins produced in this expression system are biologically active.

Oncostatin M (OSM) regulates expression of various genes in connective tissue (CT) cells, including tissue inhibitor of metalloproteinases-1 (TIMP-1). In mouse <em>fibroblast</em> cell lines MLg, NIH 3T3 and primary mouse lung <em>fibroblasts</em> (MLF), murine OSM (muOSM) stimulated high TIMP-1 mRNA expression in comparison to leukemia inhibitory <em>factor</em> (LIF), epidermal <em>growth</em> <em>factor</em> (EGF), interleukin (IL)-1beta and transforming <em>growth</em> <em>factor</em> (TGF)beta. In cell signaling, muOSM induced strong phosphorylation of extracellular-signal regulated protein kinase (Erk) 1/2, p38 and Akt in addition to phosphorylation of signal transducer and activator of transcription (STAT) 1, STAT3 and STAT5 within <em>15</em> min. LIF and TGFbeta had no such effects. EGF stimulated comparable or lower Erk1/2, p38 and Akt phosphorylation while IL-1beta induced p38 phosphorylation in the <em>fibroblast</em> cell lines. The Erk1/2 inhibitor PD98059 and the p38 inhibitor SB203580 inhibited TIMP-1 mRNA response to muOSM, whereas the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 enhanced the TIMP-1 mRNA response in NIH 3T3 and MLg cells. PD98059 and SB203580, but not LY294002, also inhibited fold induction of a chloramphenicol acetyltransferase (CAT) reporter gene driven by a minimal TIMP-1 promoter that contained a proximal activator protein-1 (AP-1) site. Co-transfection with JunB or c-Jun expression vector in NIH 3T3 cells caused marked transactivation of the TIMP-1 promoter/CAT reporter gene. muOSM caused a rapid increase of JunB and c-Jun protein in NIH 3T3 cells. PD98059 partially inhibited the increase of JunB, but not c-Jun, whereas SB203580 did not induce detectable changes in expression of either AP-1 <em>factor</em> in response to muOSM. These results demonstrate that Erk1/2 and p38 contribute to the elevation of muOSM induced TIMP-1 expression, but PI3K does not, and suggest that Erk1/2 does so by enhancing JunB expression.

We report the primary structure of MG-160, a 160 kDa membrane sialoglycoprotein residing in the medial cisternae of the Golgi apparatus of rat neurons, pheochromocytoma (PC-12), and several other cells. The cDNA encodes a polypeptide of 1,171 amino acids with an M(r) of 133,403. An intralumenal cleavable signal peptide is followed by a Pro-Gln-rich segment and 16 contiguous, approx. 60-residue-long, regularly spaced cysteine-rich segments showing sequence identities ranging from <em>15</em> to 35%. The lumenal domain is followed by a single membrane spanning domain and a short carboxy-terminal cytoplasmic tail. The protein contains 5 potential NXT glycosylation sites. The sequence of MG-160 shows no homologies with enzymes and other membrane proteins of the Golgi apparatus. MG-160 displays a so far unique feature for a membrane protein of the Golgi apparatus: namely, an upstream, open reading frame (uORF), encoding 58 amino acids, located in front of the major open reading frame (ORF). Most vertebrate mRNAs containing uORF or AUG codons in front of the major ORF encode <em>growth</em> <em>factors</em> and cell surface receptors (Geballe and Morris 1994). In that regard a 90% identity between the primary structure of MG-160 and a receptor for acidic and basic <em>fibroblast</em> <em>growth</em> <em>factors</em> (CFR), isolated from chicken embryos (Burrus et. al., 1992), may be relevant. Immunoreactivity for MG-160 has been detected in the Golgi apparatus of neural and other cells of 2-day-old chicken embryos and adult chicken; furthermore, recombinant human basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) binds MG-160 purified from rat brain. MG-160 shows no sequence similarity with members of the family of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFR) involved in signal transduction. These findings are consistent with the hypothesis that MG-160 is involved in the traffic and processing of endogenous or autocrine FGFs. This is the first example of an intrinsic membrane protein of the Golgi apparatus which binds a <em>growth</em> <em>factor</em> and may be involved in its regulation.

We recently identified a novel myristylated protein kinase C (PKC) substrate, named SSeCKS (pronounced essex), whose transcription is suppressed>> <em>15</em> fold in src- or ras-transformed rodent <em>fibroblasts</em>, but not in raf-transformed cells [1, 2]. SSeCKS associates with and controls the elaboration of a cortical cytoskeletal matrix in response to phorbol esters [2], and overexpression of SSeCKS causes <em>growth</em> arrest of untransformed NIH3T3 cells [3]. Our preliminary data suggested that SSeCKS functions as a negative mitogenic regulator by controlling cytoskeletal architecture and that serine phosphorylation of SSeCKS by kinases such as PKC alters its interaction with cytoskeletal matrices and its ability to control mitogenesis. Here, we determine the effects of culture confluency, <em>growth</em> arrest and serum response on the steady-state abundance of SSeCKS RNA and protein and on the relative level of phosphoserine-free SSeCKS. SSeCKS transcription is initially induced by serum <em>factors</em> and by contact-inhibited <em>growth</em> rather than by cell-cycle arrest induced by serum starvation, hydroxyurea or nocodazole, and following serum-induced G1/S progression, SSeCKS transcription is suppressed. SSeCKS protein is hyperphosphorylated on serine residues during G1/S progression but not during the G2/M phase. Finally, we show that the induction of SSeCKS protein expression by contact inhibition is independent of SSeCKS' serum responsiveness. These data suggest that SSeCKS expression and function can be controlled at either the transcriptional or post-translational level in response to serum <em>factors</em> and culture confluency. The data strengthen the notion that SSeCKS plays an important, yet transient, role in cell cycle progression from G0 to G1 that differs from its role in controlling contact-inhibited <em>growth</em>.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands have been recently reported to have beneficial effects on organ fibrosis. However, their effects on extracellular matrix (ECM) turnover in hypertrophic scar <em>fibroblasts</em> (HSFs), and the related molecular mechanisms are unknown. HSFs were cultured and exposed to different concentration PPAR-gamma ligands in the presence of transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1). In <em>growth</em>-arrested HSFs, a PPAR-gamma natural ligand (<em>15</em>-deoxy-D12,14-prostaglandin J2, <em>15</em>d-PGJ2) and a synthetic ligand (GW7845) dose-dependently attenuated TGFbeta1-induced expression of Connective tissue <em>growth</em> <em>factor</em> (CTGF), collagens and fibronectin. Furthermore, the suppression of CTGF mRNA and protein expression are relieved by pretreatment with an antagonist of PPAR-gamma (GW9662). Moreover, GW7845 and <em>15</em>d-PGJ2 partially inhibited the expression and phosphorylation of the TGF-beta1/Smad pathway. These results suggest that in TGFbeta1-stimulated HSFs, PPAR-gamma ligands caused an antiproliferative effect and reduced ECM production through mechanisms that included reducing CTGF expression, and a crosstalk between PPAR-gamma and Smad may be involved in the inhibitory effects of PPAR-gamma ligands.

BACKGROUND

Meniscal tears situated within the inner avascular region do not heal despite suturing. New approaches need to be developed to augment surgical repair.

OBJECTIVE

To demonstrate that basic fibroblast growth factor, used as a single agent or in combination with serum, stimulates the activity of fibrochondrocytes by enhancing proliferation and extracellular matrix synthesis in all meniscal zones, including the inner (avascular) zone of the meniscus.

METHODS

Controlled laboratory study.

METHODS

Monolayer cell cultures were prepared from the inner, middle, and outer zones of the lateral meniscus. Various concentrations of basic fibroblast growth factor were used in the presence or absence of 10% fetal calf serum. The authors measured the uptake of radiolabeled thymidine to assess cell proliferation and radioactive sulfur and proline to assess extracellular matrix formation.

RESULTS

Overall, basic fibroblast growth factor-stimulated cells from all meniscal zones to proliferate and to form new extra-cellular matrix (P <.05). The basic fibroblast growth factor (in the absence of serum) increased DNA formation and protein synthesis by cells from the inner meniscal zone by 7- and 15-fold, respectively (P <.001).

CONCLUSIONS

These results indicate that meniscal cells and, more important, cells from the avascular zone are capable of responding favorably to the addition of basic fibroblast growth factor by expressing their intrinsic potential to proliferate and generate new extracellular matrix.

CONCLUSIONS

The results suggest that it may be possible to augment surgical repair of the meniscus in the future.

The distribution of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) at the ultrastructural level in the brain of young (<em>15</em>- and 20-day-old) and adult (3-month-old) rats was investigated by immunocytochemistry. Strong staining was observed in most neurons of the cortex of young rat brain. In the same brain area of adult rat many neurons were also stained intensely, while others were negative. Neurons in the other parts of the brain and especially in the adult rat, were generally more weakly stained. The reaction product was located in the cytoplasm of the neuronal cell bodies and their processes. Astrocytes, oligodendrocytes, microglial cells, meningeal cells, choroïd epithelial cells, ependymal cells and capillary endothelial cells showed no staining.

BACKGROUND

Cigarette smoking is one of the most significant risk factors in the development and further advancement of inflammatory periodontal disease, however, the role of either nicotine or its primary metabolite cotinine in the progression of periodontitis is unclear. This study aimed to investigate the effects of nicotine and cotinine on the attachment and growth of fibroblasts derived from human periodontal ligament (PDL).

METHODS

Primary cultures were prepared from the roots of extracted premolar teeth. Cells were used at both low (P3 to P5) and high (P11 to P13) passage. Cell numbers were determined over 14 days using either the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay or with a Coulter counter. Cultures were exposed to culture medium supplemented with 1) 15% fetal calf serum (FCS) only; 2) 1% FCS only; 3) 1% FCS and nicotine (concentration range 5 ng/ml to 10 mg/ml); or 4) 1% FCS and cotinine (concentration range 0.5 ng/ml to 10 microg/ml).

RESULTS

Nicotine significantly (P <0.05, by ANOVA) inhibits attachment and growth of low passage cells at concentrations >1 mg/ml and high passage PDL fibroblasts at concentrations >0.5 mg/ml. Cotinine, at the highest concentration used (10 microg/ml), appeared to inhibit attachment and growth of both low and high passage fibroblasts but this was not statistically significant (P >0.05, by ANOVA).

CONCLUSIONS

Tobacco products inhibit attachment and growth of human PDL fibroblasts. This may partly explain the role of these substances in the progression of periodontitis.

Mutational changes coupled with endocrine, paracrine, and/or autocrine signals regulate cell division during carcinogenesis. The hormone signals remain undefined, although the absolute requirement in vitro for fetal serum indicates the necessity for a fetal serum <em>factor</em>(s) in cell proliferation. Using prostatic cancer cell (PCC) lines as a model of cancer cell proliferation, we have identified the fetal serum component activin A and its signaling through the activin receptor type II (ActRII), as necessary, although not sufficient, for PCC proliferation. Activin A induced Smad2 phosphorylation and PCC proliferation, but only in the presence of fetal bovine serum (FBS). Conversely, activin A antibodies and inhibin A suppressed FBS-induced PCC proliferation confirming activin A as one of multiple serum components required for PCC proliferation. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> was subsequently shown to synergize activin A-induced PCC proliferation. Inhibition of ActRII signaling using a blocking antibody or antisense-P decreased mature ActRII expression, Smad2 phosphorylation, and the apparent viability of PCCs and neuroblastoma cells grown in FBS. Suppression of ActRII signaling in PCC and neuroblastoma cells did not induce apoptosis as indicated by the ratio of active/inactive caspase 3 but did correlate with increased cell detachment and ADAM-<em>15</em> expression, a disintegrin whose expression is strongly correlated with prostatic metastasis. These findings indicate that ActRII signaling is required for PCC and neuroblastoma cell viability, with ActRII mediating cell fate via the regulation of cell adhesion. That ActRII signaling governs both cell viability and cell adhesion has important implications for developing therapeutic strategies to regulate cancer <em>growth</em> and metastasis.