Alzheimer's disease (AD) involves activation of apoptotic pathways that may be regulated through signaling cascades initiated by the amyloid precursor protein (APP). Enlarged endosomes have been observed in postmortem AD brains at very early stages of the disease. We show here that exogenous expression of a familial AD (FAD) mutant of APP or of the APP binding protein APP-BP1 in neurons causes enlargement of early endosomes, increased receptor-mediated endocytosis via a pathway dependent on APP-BP1 binding to APP, and apoptosis. Levels of both APP-BP1 and Rab5 are elevated in early endosomes in cortical embryonic neurons expressing APP(V642I) or APP-BP1, in cultured skin <em>fibroblast</em> cells from Down syndrome subjects, and in postmortem hippocampal tissue of individuals with AD. Indeed, Rab5 was found to bind specifically to APP-BP1, between amino acids 443 and 479. Inhibition of Rab5 or dynamin activity, but not of Eps<em>15</em> (epidermal <em>growth</em> <em>factor</em> receptor pathway substrate <em>15</em>) activity, rescued neurons from apoptosis induced by either APP(V642I) or APP-BP1, without affecting levels of intracellular or secreted amyloid-beta (Abeta). Induction of Rab5 activity via expression of a constitutively active mutant led to an increase in neuronal apoptosis more than twice that attributable to induction of endosome enlargement via a Rab5-independent mechanism, regardless of Abeta production. Together, these findings suggest that Rab5 activation via an APP/APP-BP1-initiated signaling pathway mediates neuronal apoptosis caused by FAD mutants of APP and that, within this pathway, Rab5 has a specific role in signaling that is distinct from, although not independent of, its role in trafficking.

BACKGROUND

Angiogenesis is known to be a critical and closely regulated step during bone formation and fracture healing driven by a complex interaction of various cytokines. Delays in bone healing or even nonunion might therefore be associated with altered concentrations of specific angiogenic factors. These alterations might in turn be reflected by changes in serum concentrations.

METHODS

To determine physiological time courses of angiogenic cytokines during fracture healing as well as possible changes associated with failed consolidation, we prospectively collected serum samples from patients who had sustained surgical treatment for a long bone fracture. Fifteen patients without fracture healing 4 months after surgery (nonunion group) were matched to a collective of 15 patients with successful healing (union group). Serum concentrations of angiogenin (ANG), angiopoietin 2 (Ang-2), basic fibroblast growth factor (bFGF), platelet derived growth factor AB (PDGF-AB), pleiotrophin (PTN) and vascular endothelial growth factor (VEGF) were measured using enzyme linked immunosorbent assays over a period of 24 weeks.

RESULTS

Compared to reference values of healthy uninjured controls serum concentrations of VEGF, bFGF and PDGF were increased in both groups. Peak concentrations of these cytokines were reached during early fracture healing. Serum concentrations of bFGF and PDGF-AB were significantly higher in the union group at 2 and 4 weeks after the injury when compared to the nonunion group. Serum concentrations of ANG and Ang-2 declined steadily from the first measurement in normal healing fractures, while no significant changes over time could be detected for serum concentrations of these factures in nonunion patients. PTN serum levels increased asymptotically over the entire investigation in timely fracture healing while no such increase could be detected during delayed healing.

CONCLUSIONS

We conclude that fracture healing in human subjects is accompanied by distinct changes in systemic levels of specific angiogenic factors. Significant alterations of these physiologic changes in patients developing a fracture nonunion over time could be detected as early as 2 (bFGF) and 4 weeks (PDGF-AB) after initial trauma surgery.

Administration of the antibacterial drug ampicillin (ABPC) significantly increased hepatic bile acid concentrations. In the present study, we investigated the mechanisms for the elevation of bile acid levels in ABPC-treated mice. Hepatic microsomal cholesterol 7alpha-hydroxylation and CYP7A1 mRNA level were increased 2.0-fold in ABPC-treated mice despite higher bile acid levels in the liver and small intestinal lumen. A significant change in hepatic small heterodimer partner (SHP) mRNA level was not observed in ABPC-treated mice, whereas a marked decrease in ileal <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> (FGF<em>15</em>) mRNA level was observed (3% of vehicle-treated mice). These phenomena were also observed in mice cotreated with bacitracin/streptomycin/neomycin, which are barely absorbed from the intestine. Primary bile acid contents in the small intestinal lumen were increased in ABPC-treated mice, whereas secondary bile acid, deoxycholic acid (DCA), contents were reduced to below detection limits (<0.01 micromol). In ABPC-treated mice, cotreatment with tauroDCA reversed reductions in ileal FGF<em>15</em> mRNA level. Ileal SHP mRNA level was, however, not decreased in ABPC-treated mice. ABPC administration to farnesoid X receptor (Fxr)-null mice also decreased ileal FGF<em>15</em> mRNA levels and secondary bile acid content in the small intestinal lumen. These results suggest that ABPC administration elevates hepatic primary bile acid synthesis, at least in part, through suppression of ileal FGF<em>15</em> expression.

Relationships between mevalonate and DNA synthesis were explored in quiescent human <em>fibroblasts</em> stimulated with human platelet-derived <em>growth</em> <em>factor</em> (PDGF). Studies of others have indicated that mevalonate, or a product of mevalonate other than cholesterol, is essential for DNA replication. The present studies were designed to determine whether there was a critical time in the cell cycle when mevalonate was necessary for later DNA synthesis to occur. Compactin and mevinolin, inhibitors of hydroxymethylglutaryl CoA reductase, were employed to block both the synthesis of mevalonate and of DNA. Compactin inhibited the sharp peak of DNA synthesis which occurs in cells 24 h after PDGF addition in a concentration-dependent manner. This suppression of DNA synthesis was not prevented by low density lipoprotein but was fully reversed by mevalonate. Compactin inhibited DNA synthesis when the inhibitor was present during the time interval 10-20 h after PDGF addition. Its presence only in the interval before 10 h or after 20 h had no effect. Conversely, mevalonate could fully overcome the compactin block in DNA synthesis when present during the period of from 10-20 h after PDGF addition. Mevalonate present only before 10 h or after 20 h had no effect. When mevalonate was added to mevinolin-blocked cells for the interval 10-<em>15</em> h after PDGF, the mevinolin block of DNA synthesis was 68% overcome; in contrast, only 20% of the reversal of the mevinolin block was seen when mevalonate was added from <em>15</em>-20 h. Addition of mevalonate for only the 2-h interval of from 10-12 h after PDGF overcame the mevinolin block of DNA synthesis (assayed at 24 h) by 50%. The results show that there is a critical time period, several h before S phase, when PDGF-stimulated cells require mevalonate in order for DNA synthesis to proceed at 24 h. This critical period comprised the interval of approximately 10-20 h after PDGF addition and especially the early part of this interval.

BACKGROUND

Human RNASET2 is a T2-RNase glycoprotein encoded by the RNASET2 gene, which is located on chromosome 6 (6q27). Deletion in 6q27 is associated with several human malignancies.

METHODS

A synthetic RNASET2 gene that was optimized for expression in the yeast Pichia pastoris was designed according to the cDNA sequence and was cloned under the control of the methanol-induced promoter fused to the alpha-mating secretion peptide. The recombinant protein was purified from the culture supernatant of transformed P. pastoris through an affinity Sepharose-concanavalin A column. Actin-binding activity was examined by membrane blotting using monoclonal mouse antiactin immunoglobulin M and by cross-linking in solution to G-actin using 1-[3-(dimethylamino)propyl]-3-ethyl-carboimide methiodide. The antiangiogenic activity of RNASET2 (from 0.5 microM to 10 microM) was assessed by a human umbilical vein endothelial (HUVE) cell assay in the presence of 1 microg/mL angiogenin, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), or recombinant human vascular endothelial <em>growth</em> <em>factor</em> (VEGF). Cell colony formation was examined in human colon HT29 cancer cells to assess the antitumorigenic activity of RNASET2 or the enzymatic-inactivated RNASET2 (EI-RNASET2) (1 microM each). In an athymic mouse xenograft model, LS174T human cancer cells were injected subcutaneously. When tumors were palpable, the mice were treated for 3 weeks with RNASET2 (1 mg/kg), paclitaxel (10 mg/kg or <em>15</em> mg/kg), or a combination of the 2 drugs.

RESULTS

The recombinant RNASET2 was identified as a 27-kilodalton glycoprotein that possessed the ability to bind actin in vitro. RNASET2 significantly inhibited clonogenicity in HT29 cells. EI-RNASET2 produced a similar effect, suggesting that its antitumorigenic activity is unrelated to its RNase activity. In HUVE cells, RNASET2 inhibited angiogenin-, bFGF-, and VEGF-induced tube formation in a dose-dependent manner. In athymic mice, RNASET2 inhibited the development of an LS174T-derived xenograft by 40%. A synergistic effect was obtained with combined RNASET2 and paclitaxel treatments.

CONCLUSIONS

The current results suggested that RNASET2 represents a new class of antitumorigenic and antiangiogenic drugs, and the findings of this study emphasize the advantage of using agents like RNASET2 in combined therapy.

Alcoholic liver disease (ALD) is associated with changes in the intestinal microbiota. Functional consequences of alcohol-associated dysbiosis are largely unknown. The aim of this study was to identify a mechanism of how changes in the intestinal microbiota contribute to ALD. Metagenomic sequencing of intestinal contents demonstrated that chronic ethanol feeding in mice is associated with an over-representation of bacterial genomic DNA encoding choloylglycine hydrolase, which deconjugates bile acids in the intestine. Bile acid analysis confirmed an increased amount of unconjugated bile acids in the small intestine after ethanol administration. Mediated by a lower farnesoid X receptor (FXR) activity in enterocytes, lower <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-<em>15</em> protein secretion was associated with increased hepatic cytochrome P450 enzyme (Cyp)-7a1 protein expression and circulating bile acid levels. Depletion of the commensal microbiota with nonabsorbable antibiotics attenuated hepatic Cyp7a1 expression and reduced ALD in mice, suggesting that increased bile acid synthesis is dependent on gut bacteria. To restore intestinal FXR activity, we used a pharmacological intervention with the intestine-restricted FXR agonist fexaramine, which protected mice from ethanol-induced liver injury. Whereas bile acid metabolism was only minimally altered, fexaramine treatment stabilized the gut barrier and significantly modulated hepatic genes involved in lipid metabolism. To link the beneficial metabolic effect to FGF<em>15</em>, a nontumorigenic FGF19 variant-a human FGF<em>15</em> ortholog-was overexpressed in mice using adeno-associated viruses. FGF19 treatment showed similarly beneficial metabolic effects and ameliorated alcoholic steatohepatitis.

Taken together, alcohol-associated metagenomic changes result in alterations of bile acid profiles. Targeted interventions improve bile acid-FXR-FGF<em>15</em> signaling by modulation of hepatic Cyp7a1 and lipid metabolism, and reduce ethanol-induced liver disease in mice. (Hepatology 2018;67:2<em>15</em>0-2166).

Normal human and murine <em>fibroblasts</em> can inhibit proliferation of tumor cells when cocultured in vitro. The inhibitory capacity varies depending on the donor and the site of origin of the <em>fibroblast</em>. We showed previously that effective inhibition requires formation of a morphologically intact <em>fibroblast</em> monolayer before seeding of the tumor cells. Here we show that inhibition is extended to motility of tumor cells and we dissect the <em>factors</em> responsible for these inhibitory functions. We find that inhibition is due to two different sets of molecules: (i) the extracellular matrix (ECM) and other surface proteins of the <em>fibroblasts</em>, which are responsible for contact-dependent inhibition of tumor cell proliferation; and (ii) soluble <em>factors</em> secreted by <em>fibroblasts</em> when confronted with tumor cells (confronted conditioned media, CCM) contribute to inhibition of tumor cell proliferation and motility. However, conditioned media (CM) obtained from <em>fibroblasts</em> alone (nonconfronted conditioned media, NCM) did not inhibit tumor cell proliferation and motility. In addition, quantitative PCR (Q-PCR) data show up-regulation of proinflammatory genes. Moreover, comparison of CCM and NCM with an antibody array for 507 different soluble human proteins revealed differential expression of <em>growth</em> differentiation <em>factor</em> <em>15</em>, dickkopf-related protein 1, endothelial-monocyte-activating polypeptide II, ectodysplasin A2, Galectin-3, chemokine (C-X-C motif) ligand 2, Nidogen1, urokinase, and matrix metalloproteinase 3.

The phosphaturic activity of intact, full-length, <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF-23) is well documented. FGF-23 circulates as the intact protein and as fragments generated as the result of proteolysis of the full-length protein. To assess whether short fragments of FGF-23 are phosphaturic, we compared the effect of acute, equimolar infusions of full-length FGF-23 and various FGF-23 fragments carboxyl-terminal to amino acid 176. In rats, intravenous infusions of full-length FGF-23 and FGF-23 176-251 significantly and equivalently increased fractional phosphate excretion (FE Pi) from 14 +/- 3 to 32 +/- 5% and <em>15</em> +/- 2 to 33 +/- 2% (p < 0.001), respectively. Chronic administration of FGF-23 176-251 reduced serum Pi and serum concentrations of 1alpha,25-dihydroxyvitamin D. Shorter forms of FGF-23 (FGF-23 180-251 and FGF-23 184-251) retained phosphaturic activity. Further shortening of the FGF-23 carboxyl-terminal domain, however, abolished phosphaturic activity, as infusion of FGF-23 206-251 did not increase urinary phosphate excretion. Infusion of a short fragment of the FGF-23 molecule, FGF-23 180-205, significantly increased FE Pi in rats and reduced serum Pi in hyperphosphatemic Fgf-23 ( -/- ) knockout mice. The activity of FGF-23 180-251 was confirmed in opossum kidney cells in which the peptide reduced Na(+)-dependent Pi uptake and enhanced internalization of the Na(+)-Pi IIa co-transporter. We conclude that carboxyl terminal fragments of FGF-23 are phosphaturic and that a short, 26-amino acid fragment of FGF-23 retains significant phosphaturic activity.

About 600,000 people in the United States are estimated to be affected by venous ulcers. The cornerstone of care of chronic venous ulcers involves the application of compression bandages. Other therapies include treatment of associated infection, treatment for edema and inflammation, and debridement when necessary. Repifermin, a recombinant human KGF-2 (<em>fibroblast</em> <em>growth</em> <em>factor</em>-10), exerts a proliferative effect on epithelial cells, in vitro and in vivo, and has been shown to accelerate wound healing in several experimental animal models. A randomized, double-blind, parallel-group, placebo-controlled, multicenter study was conducted to evaluate the safety and efficacy of topical repifermin treatment, for 12 weeks, in the healing of chronic venous ulcers in 94 patients. Repifermin was shown to accelerate wound healing, with significantly more patients achieving 75% wound closure with repifermin than with placebo. The treatment effect appeared more marked for a subgroup of patients with initial wound areas < or = <em>15</em> cm2 and wound ages of < or = 18 months. A longer duration of treatment (e.g., 26 weeks) may allow better differentiation of the benefit of repifermin compared with placebo, particularly with respect to complete wound closure. The safety assessment showed that repifermin was well tolerated.

A continuous cell line, the flounder embryonic cell line (FEC), was established from gastrula-stage embryos of a marine cultured fish, the Japanese flounder Paralichthys olivaceus and cultured for more than 200 d with more than 60 passages. FEC cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with antibiotics, fetal bovine serum (FBS), sea perch serum (SPS), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). The cells were small and round, and grew actively and stably in culture. The effect of temperature, FBS concentration and bFGF on FEC cell <em>growth</em> was examined. Cells grew well between 24 and 30 degrees C, but had a reduced <em>growth</em> rate below 18 degrees C. The <em>growth</em> rate of FEC cells in medium containing <em>15</em>% FBS was higher than that in medium containing 7.5% FBS. Addition of bFGF to the medium also significantly increased the <em>growth</em> rate. Chromosome analysis revealed that FEC cells have a normal diploid karyotype with 2n = 48. High survival rate was obtained after cryopreservation of cell cultures. The susceptibility of the cell line to piscine viruses was examined. Two viruses tested were shown to induce CPE (cytopathic effect) on FEC cells. FEC cell culture infected with fish iridovirus was further elucidated by electron microscopy. Many virus particles were found in the cytoplasm of the virus-infected FEC cells. These results indicated that the FEC cell line could be potentially used to isolate and study fish viruses.

Muscle <em>growth</em> during embryogenesis is the result of a balance between the proliferation of myoblasts and their differentiation into mature, contractile fibers. <em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) are potent stimulators of myoblast proliferation and inhibitors of myoblast differentiation in vitro. However, it is not clear at present if FGFs and their receptors regulate this process in vivo, partially because no FGF receptor was known to be expressed by myoblasts during embryogenesis. In this study, we have used quail/chick grafting and BrdU labeling techniques to demonstrate that a recently cloned avian FGF receptor, FREK, is expressed by replicating skeletal muscle myoblasts, while differentiated muscle cells no longer express this receptor. In the limb, muscle progenitors originating from the somite start expressing FREK at 3 days of development (E3). FREK expression in the limb myoblasts follows that of Pax-3 and Pax-7, but precedes that of MyoD. Since MyoD expression signals the onset of terminal differentiation, this demonstrates that FREK is expressed in muscle progenitors prior to overt muscle differentiation. A more complex situation is observed in the trunk region, where a first wave of MyoD-positive myocytes, which are postmitotic and never express FREK, appear in the early myotomal compartment of the somite. Slightly later, at E2.5, FREK-positive myoblasts migrate into the myotome as a second wave of muscle progenitors, <em>15</em> hr after the first MyoD-positive cells. FREK's expression by myoblasts arising at all stages of myogenesis indicates that this <em>growth</em> <em>factor</em> receptor represents one of the earliest molecular markers for this cell population. FREK's prominent expression during muscle differentiation sets it apart from other FGF receptors and suggests that this molecule plays an important role during muscle <em>growth</em> and differentiation.

Signaling by the Caenorhabditis elegans <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor EGL-<em>15</em> is activated by LET-756, a <em>fibroblast</em> <em>growth</em> <em>factor</em>, and attenuated by CLR-1, a receptor tyrosine phosphatase. Hyperactive EGL-<em>15</em> signaling results in a dramatic Clr phenotype characterized by the accumulation of clear fluid within the pseudocoelomic space, suggesting that regulated EGL-<em>15</em> signaling is essential for fluid homeostasis in C. elegans. To determine the cellular focus of EGL-<em>15</em> signaling, we identified an enhancer element (e<em>15</em>) within the egl-<em>15</em> promoter, which is both necessary for the promoter activity and sufficient when duplicated to drive either egl-<em>15</em> or clr-1 rescue activity. This enhancer drives GFP expression in hypodermal cells. Consistent with this finding, immunofluorescence studies of EGL-<em>15</em> indicate that EGL-<em>15</em> is expressed in hypodermal cells, and hypodermal promoters can drive full clr-1 and egl-<em>15</em> rescue activity. Moreover, a mosaic analysis of mpk-1, which acts downstream of egl-<em>15</em>, suggests that its suppression of Clr (Soc) function is required in the hypodermis. These results suggest that EGL-<em>15</em> and CLR-1 act in the hypodermis to regulate fluid homeostasis in worms.

Okadaic acid, a phosphatase inhibitor from a marine organism, mimics tumor necrosis <em>factor</em>/interleukin-1 (TNF/IL-1) in inducing changes in early cellular protein phosphorylation. A total of approximately 116 proteins exhibit significant and concordant changes in phosphorylation or dephosphorylation within <em>15</em> min in human <em>fibroblasts</em> activated by either okadaic acid, TNF, or IL-1. The fidelity of this mimicry by okadaic acid extends to the phosphorylation of the 27 hsp complex, stathmin, eIF-4E, myosin light chain, nucleolin, epidermal <em>growth</em> <em>factor</em> receptor, and other cdc2-kinase substrates (c-abl, RB, and p53). The okadaic acid-induced pattern of protein phosphorylation is distinct from that observed in cells treated with phorbol 12-myristate 13-acetate or with ligands like epidermal <em>growth</em> <em>factor</em>, cyclic AMP agonists, bradykinin, or interferons. Like TNF, okadaic acid also induces the transcription of immediate early response genes like c-jun and Egr-1 as well as the interleukin-6 genes. The overall early effects of okadaic acid uniquely parallel those of TNF/IL-1 and not those of other cytokines or ligands. Regulation of protein phosphatase inhibition is discussed as a mechanism for TNF/IL-1 signal transduction.

Stromal cells can dramatically affect the <em>growth</em> and metastatic capability of breast carcinoma cells. <em>Growth</em> <em>factors</em>, considered to be important mediators of this process, act as either mitogenic or mito-inhibitory regulators. We have developed an in vitro coculture system to examine the influence of adipocytes, a dominant mammary stromal cell type, on the <em>growth</em> of a murine mammary carcinoma, SP1. Previously, we have reported that conditioned medium (CM) from 3T3-L1 adipocytes can promote in vitro <em>growth</em> of SP1 cells. We now show that the major mitogenic signal derived from 3T3-L1 adipocyte CM is mediated by hepatocyte <em>growth</em> <em>factor</em> (HGF). Neutralizing antibody against HGF at <em>15</em> micrograms/ml completely abrogated mitogenic activity of 3T3-L1 CM. Furthermore, heparin, an inhibitor of biological activity of HGF, inhibited the mitogenic activity of 3T3-L1 CM. Western blot analysis also confirmed the presence of HGF in 3T3-L1 CM. Although basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and insulin-like <em>growth</em> <em>factor</em> I (IGF-I) were mitogenic for SP1 cells, neutralizing antibodies against IGF-I, bFGF, platelet-derived <em>growth</em> <em>factor</em> (PDGF), and epidermal <em>growth</em> <em>factor</em> (EGF) did not inhibit the mitogenic activity of 3T3-L1 CM. Immunoprecipitation and immunoblotting of HGF receptor/c-met showed that c-met is expressed at high level in SP1 cells, and is phosphorylated following HGF ligation. Together, our present data demonstrate that 3T3-L1 adipocytes secrete HGF, which stimulates SP1 cell <em>growth</em> by a paracrine mechanism. Furthermore, the mitogenic effect of 3T3-L1 CM requires HGF receptor ligation and activation of tyrosine kinase signaling cascades in SP1 cells. These results highlight the importance of stromal-tumor cell interactions and suggest that HGF secreted by adipocytes may be a key regulator of mammary tumor <em>growth</em>.

OBJECTIVE

Disruption of stromal-epithelial interactions favoring prostate cancer progression may affect the phenotype of the disease. We did a preoperative study to test the hypothesis that thalidomide, an active agent in metastatic disease, is a modulator of the tumor microenvironment.

METHODS

Eighteen men with high-risk prostate cancer were given thalidomide at doses escalated to 600 mg for 12 weeks, followed by radical prostatectomy. We constructed tissue microarrays from prostatectomy specimens from <em>15</em> treated patients and <em>15</em> matched untreated control subjects to assess effects of thalidomide on the tumor microenvironment. We compared the immunohistochemical expression of three groups of markers linked to angiogenesis, stromal-epithelial interactions, or the epithelial compartment. Levels of circulating basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, interleukin-6, tumor necrosis <em>factor</em>-alpha, and vascular endothelial <em>growth</em> <em>factor</em> were also assessed.

RESULTS

Thalidomide was well tolerated and induced a median reduction in prostate-specific antigen of 41% without affecting testosterone. Tissue microarray analyses indicated modulation of vascular marker expression accompanied by a reduction in microvessel density in the treated group. Comparison of broader stromal-epithelial interaction markers between treated and control groups suggested a transition to a less aggressive phenotype as a result of thalidomide treatment. Hedgehog signaling was attenuated and the ratio of matrix metalloproteinases to E-cadherin shifted to favor E-cadherin. No differences were noted in proliferation or apoptosis in the epithelial compartment.

CONCLUSIONS

These findings are the first clinical evidence to support the hypothesis that the reported thalidomide clinical efficacy is attributable to early modulation of the tumor microenvironment and suggest that stromal-targeting therapies will be effective against prostate cancer.

In this study we describe the activation of a protein kinase which phosphorylates a peptide, T669, comprising amino acids 663-681 of the epidermal <em>growth</em> <em>factor</em> receptor and containing the phosphate acceptor site Pro-Leu-Thr669-Pro. In the human epidermoid carcinoma cell line KB, T669 kinase activity in cytosolic extracts peaked (up to <em>15</em>-fold compared with basal levels) <em>15</em>-30 min after addition of interleukin-1 (IL-1) and closely paralleled receptor occupancy with a half-maximally effective concentration of approximately 100 pM IL-1 alpha. IL-1 treatment elevated T669 kinase activity to a variable extent in selected <em>fibroblast</em> lines, the hepatoma cell line HepG2, and the murine thymoma EL4 6.1. An IL-1 receptor-negative EL4 variant and the B cell lines 70Z/3, CB23, and RPMI 1788 did not respond in this way. All of the cell lines except 70Z/3 showed increased levels of T669 kinase when treated with the protein kinase C activator phorbol myristate acetate and/or with epidermal <em>growth</em> <em>factor</em>. This finding is in agreement with a previous study (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Activators of protein kinase A did not mimic the ability of IL-1 to stimulate T669 kinase activity, nor did the protein kinase C inhibitor staurosporine abrogate the effect of IL-1. T669 kinase activity from IL-1-stimulated KB cells was partially purified by ion exchange, hydrophobic interaction, and size exclusion chromatography. The partially purified enzyme phosphorylated myelin basic protein, a characteristic substrate of microtubule-associated protein-2 kinase (MAP-2 kinase) and the peptide Arg-Arg-Arg-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4 from RNA polymerase II. Western blotting of chromatographic fractions revealed that T669 kinase activity corresponded with two proteins of 43 and 45 kilodaltons which cross-reacted with antibodies raised against peptide sequences of rat extracellular signal-regulated kinase-1/microtubule-associated protein-2 kinase. T669 kinase activity was critically dependent on the presence of phosphatase inhibitors. Since both the 43- and 45-kDa proteins, immunoprecipitated from [32P]phosphate-labeled cells, demonstrated a dramatic increase in their levels of serine, threonine, and tyrosine phosphorylation after brief treatment with IL-1, we conclude that IL-1 modulates the activity of these extracellular signal-regulated kinase/microtubule-associated protein-2 kinases by altering the level of their phosphorylation.

The secondary and tertiary structure of recombinant human acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and <em>15</em>% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi-empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this <em>growth</em> <em>factor</em> in vivo.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGF) constitute a family of at least 9 members which act through high-affinity tyrosine-kinase receptors encoded by 4 distinct genes. In humans, the FGFR1 gene is located in chromosomal region 8p12. Its amplification and expression were examined in a panel of 110 breast carcinoma samples by Southern- and Northern-blot analyses. FGFR1 was amplified in 9% and overexpressed in about <em>15</em>% of the tumors studied. In situ hybridization experiments were performed on tissue sections of normal breast and tumors with a high level of FGFR1 expression. In both normal and tumoral tissues, FGFR1 RNA was detected in the epithelial cells. Overexpression of FGFR1 seems to be associated with small, well-differentiated diploid tumors.

Transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1), which appears in high concentrations in fibrotic cardiac tissue, is a potent inductor of tissue collagen deposition and of the differentiation of <em>fibroblasts</em> to myo<em>fibroblasts</em>. It is accepted that TGF-beta 1 is a potent stimulator of collagen secretion by <em>fibroblasts</em>. The aim of the present study was to determine which type of cells, <em>fibroblasts</em> and/or myo<em>fibroblasts</em> are stimulated, in terms of collagen production, by TGF-beta 1. Therefore, using cultures of second-passage rat cardiac <em>fibroblasts</em>, we investigated the dose- (0.003-<em>15</em> ng/ml) and time-dependence (2-48 h) of the TGF-beta 1-induced effects on collagen production and on the appearance of myo<em>fibroblasts</em>, as estimated by the presence of alpha-smooth muscle actin (alpha-SMA; a marker of myo<em>fibroblasts</em>). The reversibility of the TGF-beta 1-stimulated effects was also studied. The dose- and time-dependent stimulation of collagen production was closely associated with the induction of alpha-SMA. TGF-beta 1 did not change the cell phenotype or increase collagen production in rat cardiac <em>fibroblasts</em> cultures after a long incubation (24-28 h) at low concentrations (< 1 ng/ml), or after a short incubation (2-4 h) at high concentrations (1-<em>15</em> ng/ml). However, after a long incubation at high concentrations, TGF-beta 1 changed the cell phenotype and increased collagen production in these cultures through the differentiation of <em>fibroblasts</em> to myo<em>fibroblasts</em>. A maximal increase of collagen production (two-fold, p < 0.001) was observed after incubation of <em>fibroblasts</em> with <em>15</em> ng/ml TGF-beta 1 for 48 h. Under these conditions, alpha-SMA was increased by 3.5-fold (p < 0.001) and second-passage cultures of <em>fibroblasts</em> and their offspring in the next passage consisted mainly of myo<em>fibroblasts</em>. The stimulation of collagen by <em>15</em> ng/ml TGF-beta 1 for 48 h was irreversible. In fact, additional incubation of these second-passage TGF-beta 1-stimulated cultures without TGF-beta 1 for 2 days did not decrease the high activity of collagen production. Moreover, the third-passage offspring of these TGF-beta 1-stimulated <em>fibroblasts</em> cultured without TGF-beta 1 also showed a higher production of collagen compared with control <em>fibroblasts</em>. Furthermore, the increased collagen production in the third-passage <em>fibroblast</em> offspring of the second-passage TGF-beta 1-stimulated <em>fibroblasts</em> could not be further stimulated by TGF-beta 1. Thus, the activity of collagen production in TGF-beta 1-stimulated cultures and in their next passage offspring is not sensitive to TGF-beta 1. Our data suggest that TGF-beta 1-stimulated collagen production in cultures of adult rat cardiac ventricular <em>fibroblasts</em> cannot be explained by a direct stimulation of collagen production, either in <em>fibroblasts</em> or in myo<em>fibroblasts</em>. Instead, TGF-beta 1 induces differentiation of <em>fibroblasts</em> to myo<em>fibroblasts</em>, the latter having a higher activity for collagen production than the former.

Current concepts suggest that macrophages may play a central role in pulmonary fibrosis by virtue of their ability to release a variety of cytokines. In this study, the expression of interleukin (IL)-1 alpha and beta, platelet-derived <em>growth</em> <em>factor</em> (PDGF) A and B, and insulin-like <em>growth</em> <em>factor</em> (IGF) I in BAL cells, which may be involved in <em>fibroblast</em> proliferation, was investigated in murine bleomycin (BLM)-induced pulmonary fibrosis. BAL cells were obtained at 1, <em>15</em>, and 29 days from Institute for Cancer Research mice after 10 days of intraperitoneal administration of BLM. The relative amounts of cytokine messenger RNA (mRNA) were evaluated by the reverse transcription-polymerase chain reaction method, which simultaneously amplified complementary DNA for cytokines and beta-actin as an internal control. The level of IL-1 beta mRNA in BLM-treated mice was increased 4.5-fold compared with that in saline solution-treated (control) mice 1 day after treatment, while no significant differences were observed between the two groups at <em>15</em> and 29 days. The mRNAs of PDGF-A and IGF-I in BLM-treated mice were sustained at levels eightfold and threefold to fourfold, respectively, those of controls over 4 weeks. No significant differences were noted in IL-1 alpha and PDGF-B expression between the two groups. We conclude that IL-1 beta released from macrophages may be important in the early phase of inflammatory responses and that PDGF-A and IGF-I may play important roles in the development of BLM-induced pulmonary fibrosis.

Non-syndromic cleft lip and palate (NS CLP) is a complex birth defect resulting from multiple genetic and environmental <em>factors</em>. We have previously reported the sequencing of the coding region of genes in the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) signaling pathway, in which missense and non-sense mutations contribute to approximately 5%-6% NS CLP cases. In this article we report the sequencing of conserved non-coding elements (CNEs) in and around 11 of the FGF and FGFR genes, which identified 55 novel variants. Seven of variants are highly conserved among>>/=8 species and 31 variants alter transcription <em>factor</em> binding sites, 8 of which are important for craniofacial development. Additionally, <em>15</em> NS CLP patients had a combination of coding mutations and CNE variants, suggesting that an accumulation of variants in the FGF signaling pathway may contribute to clefting.

Abstract Platelet derivatives are commonly used in wound healing and tissue regeneration. Different procedures of platelet preparation may differentially affect <em>growth</em> <em>factor</em> release and cell <em>growth</em>. Preparation of platelet-rich fibrin (PRF) is accompanied by release of <em>growth</em> <em>factors</em>, including platelet-derived <em>growth</em> <em>factor</em> (PDGF), vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and transforming <em>growth</em> <em>factor</em> β1 (TGFβ1), and several cytokines. When compared with the standard procedure for platelet-rich plasma (PRP), PRF released 2-fold less PDGF, but>><em>15</em>-fold and >2-fold VEGF and TGFβ1, respectively. Also, the release of several cytokines (IL-4, IL-6, IL-8, IL-10, IFNγ, MIP-1α, MIP-1β and TNFα) was significantly increased in PRF-conditioned medium (CM), compared to PRP-CM. Incubation of both human skin <em>fibroblasts</em> and human umbilical vein endothelial cells (HUVECs) with PRF-derived membrane (mPRF) or with PRF-CM enhanced cell proliferation by >2-fold (p<0.05). Interestingly, PRP elicited <em>fibroblast</em> <em>growth</em> at a higher extent compared to PRF. At variance, PRF effect on HUVEC <em>growth</em> was significantly greater than that of PRP, consistent with a higher concentration of VEGF in the PRF-CM. Thus, the procedure of PRP preparation leads to a larger release of PDGF, as a possible result of platelet degranulation, while PRF enhances the release of proangiogenic <em>factors</em>.

The roles of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF [FGF-2]) in early postnatal regulation of coronary angiogenesis were investigated by administering neutralizing antibodies to these <em>growth</em> <em>factors</em> between postnatal days 5 and 12. Immunohistochemistry and Western blotting both revealed decreases in VEGF protein in the hearts of rats treated with either antibody. In contrast, bFGF mRNA increased in both treated groups, whereas VEGF mRNA was unchanged. Using stereological assessment of perfusion-fixed hearts, we found that both anti-VEGF and anti-bFGF inhibited the rapid and marked capillary <em>growth</em> that occurs during this time period and that the effects of the two neutralizing antibodies are not additive. Arteriolar <em>growth</em>, as indicated by a lower length density, was inhibited by anti-bFGF, but not anti-VEGF. When both anti-VEGF and anti-bFGF were administered, arteriolar length density was not significantly lower, but the population of small arterioles ((<em>15</em> microm) was markedly reduced, whereas the percentage of large arterioles (26 to 50 microm) more than doubled. Thus, inhibition of both <em>growth</em> <em>factors</em> negated or limited the formation of small arterioles and facilitated an expansion of the largest arterioles. These in vivo data are the first to document that during the early neonatal period, (1) both VEGF and bFGF modulate capillary <em>growth</em>, (2) bFGF facilitates arteriolar <em>growth</em>, and (3) the two <em>growth</em> <em>factors</em> interact to establish the normal hierarchy of the arteriolar tree.

Tenascin C (TNC) is an extracellular matrix protein that is upregulated during development as well as tissue remodeling. TNC is comprised of multiple independent folding domains, including <em>15</em> fibronectin type III-like (TNCIII) domains. The fifth TNCIII domain (TNCIII5) has previously been shown to bind heparin. Our group has shown that the heparin-binding fibronectin type III domains of fibronectin (FNIII), specifically FNIII12-14, possess affinity towards a large number of <em>growth</em> <em>factors</em>. Here, we show that TNCIII5 binds <em>growth</em> <em>factors</em> promiscuously and with high affinity. We produced recombinant fragments of TNC representing the first five TNCIII repeats (TNCIII1-5), as well as subdomains, including TNCIII5, to study interactions with various <em>growth</em> <em>factors</em>. Multiple <em>growth</em> <em>factors</em> of the platelet-derived <em>growth</em> <em>factor</em> (PDGF) family, the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family, the transforming <em>growth</em> <em>factor</em> beta (TGF-β) superfamily, the insulin-like <em>growth</em> <em>factor</em> binding proteins (IGF-BPs), and neurotrophins were found to bind with high affinity to this region of TNC, specifically to TNCIII5. Surface plasmon resonance was performed to analyze the kinetics of binding of TNCIII1-5 with TGF-β1, PDGF-BB, NT-3, and FGF-2. The promiscuous yet high affinity of TNC for a wide array of <em>growth</em> <em>factors</em>, mediated mainly by TNCIII5, may play a role in multiple physiological and pathological processes involving TNC.