Skeletal cells secrete insulin-like <em>growth</em> <em>factors</em> (IGFs) I and II and six known IGF binding proteins (IGFBPs). IGFBP-5 stimulates bone formation, and its synthesis correlates with changes in osteoblast cell <em>growth</em>. We tested the effects of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), transforming <em>growth</em> <em>factor</em> beta 1 (TGF beta 1), and platelet-derived <em>growth</em> <em>factor</em> (PDGF) BB on IGFBP-5 expression in cultures of osteoblast-enriched cells from <em>22</em>-day-old fetal rat calvariae (Ob cells). Treatment of Ob cells with bFGF, TGF beta 1, and PDGF BB caused a time- and dose-dependent decrease in IGFBP-5 mRNA levels and inhibited IGFBP-5 polypeptide levels in the extracellular matrix. The effects of bFGF, TGF beta 1, and PDGF BB on IGFBP-5 transcripts were independent of cell division and were observed in the presence and absence of hydroxyurea. bFGF, TGF beta 1, and PDGF BB did not modify the decay of IGFBP-5 mRNA in transcriptionally arrested Ob cells, and they inhibited IGFBP-5 heterogeneous nuclear RNA and the rate of IGFBP-5 transcription. In conclusion, bFGF, TGF beta 1, and PDGF BB inhibit IGFBP-5 expression in Ob cells independently of their mitogenic activity and through mechanisms that involve decreased transcription.

Okadaic acid (OKA), a potent inhibitor of serine phosphatases at concentrations as low as 20-25 nM, induces apoptosis of R- mouse embryo <em>fibroblasts</em>, which are 3T3-like cells devoid of type 1 insulin-like <em>growth</em> <em>factor</em> receptors (IGF-IRs). From R- cells, we have generated (by stable transfection) cell lines with IGF-IR numbers ranging from 0 (R- cells) to >10(6) receptors per cell. The wild-type IGF-IR protects R- cells from OKA-induced apoptosis, its protective effect being exquisitely dependent on the number of receptors. A small increment in wild-type receptor number (from 15 x 10(3) to <em>22</em> x 10(3) receptors/cell) is sufficient to change R(-)-derived cells from sensitive to resistant to apoptosis. We have also studied the effect of various mutations of the IGF-IR on its ability to protect R(-)-derived cells from OKA-induced apoptosis. Our data indicate a correlation between protection from apoptosis and the ability of the receptor to respond to insulin-like <em>growth</em> <em>factor</em> I with mitogenesis.

OBJECTIVE

To determine whether adrenomedullin (ADM), a vasorelaxant peptide is produced and secreted by human retinal pigment epithelial (RPE) cells, whether ADM expression is regulated by inflammatory cytokines and a growth factor, and whether ADM has proliferative effects on these cells.

METHODS

Production and secretion of ADM by cultured human RPE cells were examined by Northern blot analysis and radioimmunoassay. Regulation of the ADM expression by basic fibroblast growth factor, interferon (IFN)-gamma, tumor necrosis factor-alpha, interleukin (IL)1beta, or all-trans-retinoic acid was studied. In addition, proliferative effects of ADM on human RPE cells were examined by modified 3-(4,5-dimetylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay.

RESULTS

ADM mRNA was expressed constitutively in all three human RPE cell lines (F-0202, D407, and ARPE-19) examined. Immunoreactive ADM was detected in the cultured media by radioimmunoassay. Sephadex G-50 column chromatography of the cultured medium showed a single peak eluting in the position of ADM-(1-52). Treatment with IFN-gamma or IL-beta increased ADM mRNA levels and immunoreactive-ADM levels in the medium in dose- and time-dependent manners in ARPE-19 cells. Exogenously added ADM increased the number of F-0202 cells and ARPE-19 cells, and the treatment with ADM antibody or ADM-(22-52) (an ADM antagonist) decreased it.

CONCLUSIONS

Human RPE cells produced and secreted ADM. IFN-gamma and IL-1beta induced ADM expression in ARPE-19 cells. Furthermore, ADM stimulated proliferation of RPE cells. These results raise the possibility that ADM is related to the pathophysiology of some inflammatory and proliferative ocular diseases.

This study evaluated the effects of basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, transforming <em>growth</em> <em>factor</em>-beta 1, insulin-like <em>growth</em> <em>factor</em>-1, and insulin on the incorporation of thymidine and sulfate in human osteoarthritic articular cartilage. Tissue explants were obtained from 11 patients undergoing total knee arthroplasty and were categorized as nonfibrillated or fibrillated cartilage. The explants were cultured for <em>22</em> days, with changes of medium and <em>growth</em> <em>factor</em> every 72 hours, and labeled with [3H]thymidine and [35S]sulfate. <em>Growth</em> <em>factors</em> were used in the following concentrations: basic <em>fibroblast</em> <em>growth</em> <em>factor</em> at 1, 10, and 100 ng/ml; transforming <em>growth</em> <em>factor</em>-beta 1 at 0.5, 5, and 50 ng/ml; insulin-like <em>growth</em> <em>factor</em>-1 at 0.15, 1.5, and 15 ng/ml; and insulin at 0.05, 0.5, and 5 micrograms/ml. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> decreased thymidine incorporation to 70% and sulfate incorporation to less than 20% that of the <em>growth</em> <em>factor</em>-free controls. Transforming <em>growth</em> <em>factor</em>-beta 1 had no significant effect on thymidine incorporation, whereas the concentrations studied inhibited sulfate incorporation to approximately 40% that of the controls. At the concentrations tested, insulin-like <em>growth</em> <em>factor</em>-1 had no significant effect on incorporation of either thymidine or sulfate. In contrast, insulin significantly stimulated the incorporation of both. Compared with <em>growth</em> <em>factor</em>-free controls, insulin maximally increased thymidine incorporation by a <em>factor</em> (+/- SEM) of 2.36 +/- 0.47 and 1.69 +/- 0.19 in nonfibrillated and fibrillated explants, respectively; sulfate incorporation was maximally increased 1.60 +/- 0.24 and 1.92 +/- 0.29-fold for nonfibrillated and fibrillated explants, respectively. Of the <em>factors</em> tested, insulin demonstrated the greatest promise for promoting a synthetic response that may contribute to the regeneration of osteoarthritic cartilage.

Calcineurin homologous protein 1 (CHP1) is a widely expressed, <em>22</em>-kDa myristoylated EF-hand Ca(2+)-binding protein that shares a high degree of similarity with the regulatory B subunit of calcineurin (65%) and with calmodulin (59%). CHP1 localizes to the plasma membrane, the Golgi apparatus, and the nucleus and functions to regulate trafficking of early secretory vesicles, activation of T cells, and expression and transport of the Na-H exchanger NHE1. Although CHP1 contains nuclear export signals, whether its nuclear and cytoplasmic localization is regulated and has distinct functions remain unknown. We show that CHP1 is predominantly in the nucleus in quiescent <em>fibroblasts</em>, is translocated to cytoplasmic compartments with <em>growth</em> medium, and that translocation is inhibited by mutations in the nuclear export motifs. In a screen for proteins co-precipitating with CHP1 in quiescent cells we identified the upstream binding <em>factor</em> UBF, a DNA-binding protein and component of the RNA polymerase I complex regulating RNA synthesis. The CHP1-UBF interaction is restricted to the nucleus and inhibited by Ca(2+). Nuclear retention of CHP1 attenuates the abundance of UBF in the nucleolus and inhibits RNA synthesis when quiescent cells are transferred to <em>growth</em> medium. These data show UBF as a newly identified CHP1-binding protein and regulation of RNA synthesis as a newly identified function for nuclear-localized CHP1, which is distinct from CHP1 functions in the cytosol.

Human neonatal <em>fibroblasts</em> were cultured on a lactate-glycollate copolymer scaffold for 12-16 days to form a three-dimensional dermal equivalent tissue. The cellular content of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) mRNA in these three-dimensional cultures was <em>22</em>-fold greater than that observed in the same <em>fibroblasts</em> grown as monolayers. No induction was shown by hepatocyte <em>growth</em> <em>factor</em> (HGF) or angiopoietin 1 indicating that the effect was specific to VEGF. The predominant VEGF splice variant, detected by RT-PCR corresponded to the 121 amino acid form, with less of the 165 amino acid form. The cell-associated forms (189 and 206 amino acids) comprised less than 1% of the total VEGF mRNA. VEGF and HGF proteins, determined by ELISA, were secreted in physiologically significant amounts, 0.5-4 ng per 24 h/10(6) cells. Conditioned medium from the three-dimensional cultures stimulated proliferation of endothelial cells in a dose-dependent manner and induced cellular expression of integrin alpha(v)beta(3). Conditioned medium from the same dermal <em>fibroblasts</em> cultured in monolayer showed little angiogenic activity in any of these assays. Using the chorioallantoic membrane (CAM) angiogenesis assay, the cultures stimulated blood vessel production 2.8-fold over scaffold alone. VEGF-neutralizing antibody reduced the vessel development in the CAM to the level in the scaffold control. Anti-HGF antibody had no significant effect. In conclusion, three-dimensional cultures of dermal equivalent tissue express angiogenic activity to a greater extent than monolayer cultures, some of which can be assigned to VEGF.

<em>Growth</em> <em>factors</em> coordinately regulate a variety of genes associated with pathological states including tumor invasion and metastasis. Overexpressed epidermal <em>growth</em> <em>factor</em> receptor (EGFR) on tumor cell surfaces is associated with enhanced cell attachment and migration into extracellular matrices, which promotes tumor aggressiveness. We have demonstrated that epidermal <em>growth</em> <em>factor</em> (EGF) up-regulates the cell surface adhesion molecule CD44 at both the mRNA and protein levels on mouse <em>fibroblasts</em> expressing full-length wild-type EGFR (NR6-WT) but not on EGFR-deficient cells (NR6-P). This increases cell attachment to hyaluronic acid. In this investigation, transcriptional regulation of CD44 by EGF was confirmed by defining an EGF-regulatory element. By employing human CD44 gene promoter-chloramphenicol acetyltransferase (CAT) constructs transfected into NR6-WT cells, EGF inducibility was observed within a 120-base pair (bp) DNA fragment located 450 bp upstream of the RNA initiation site. Differential EGF inducibility was found among different cell lines chosen, indicating a 3.2- and 1.8-fold enhancement in DU145 cells carrying exogenous wild-type EGFR and in MCF-7 cells, respectively, while minimal EGF induction was found in cervical cancer HeLa cells. Utilizing gel shift assays, a time-dependent increase of DNA-protein complex formation was found upon EGF stimulation in NR6-WT cells but not in NR6-P cells. Based upon these observations, a novel <em>22</em>-bp EGF regulatory element (ERE) (5'--604CCCTCTCTCCAGCTCCTCTCCC-583-3') was isolated from the CD44 gene promoter. This ERE conferred DNA-protein binding ability in vitro, as well as the full functional recovery of EGF inducibility of CAT activity when linked to a homologous CD44 promoter or a SV40 promoter driving a CAT reporter gene. A two-base mutation of the ERE completely eliminated its binding activity as well as its EGF inducibility of CAT expression. Our studies indicate that EGF induces CD44 gene expression through an interaction between a specific ERE and putative novel transcriptional <em>factor</em> so as to regulate cell attachment to extracellular matrix.

As an adjuvant therapy for patients with high risk of recurrent melanoma, high-dose interferon (IFN)-alpha2b therapy has been shown to have some efficacy. We examined <em>22</em> patients with resected melanoma who were treated with repeated injections of recombinant IFN-alpha2b during the treatment. Both angiogenic and immune parameters were measured. White blood cells (WBCs) and lymphocyte numbers, lymphocyte subpopulations, serum concentrations of IFN-alpha and anti-IFN-alpha antibodies, and the serum vascular endothelial <em>growth</em> <em>factor</em> (VEGF), interleukin (IL)-8, and basis <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) concentrations were determined over time in resected, recurrence-free patients with American Joint Committee on Cancer (AJCC) stage III melanoma with one or less (LN+ < or = 1, n = 7) or more than one (LN+>> 1, n = 8) lymph nodes involved, and AJCC stage IV resected disease (n = 7). Follow-up and recurrence-free intervals were longer in stage III (LN+ < or = 1) patients compared with stage IV patients (P < 0.05). The number of WBCs and lymphocytes decreased during the treatment for all patient groups (P < 0.001). In addition, percentages of CD8 and CD20 were higher in stage IV patients than in stage III (LN+>> 1) and stage III (LN+ < or = 1) patients at the beginning of therapy (P < 0.05). A significant increase in the percentage of CD20+ cells, mostly B lymphocytes, was observed in the stage III (LN+>> 1) and stage III (LN+ < or = 1) patients over time but not in stage IV patients (P < 0.001). Low IL-8 and bFGF concentrations at the beginning of therapy were associated with significantly longer recurrence-free survival (P < 0.05). These results warrant a larger trial to determine if the differences observed in patients before treatment can provide prognostic markers in patients receiving IFN-alpha2b therapy.

Normal Rat-1 <em>fibroblasts</em> and Rat-1 cells transformed by the H-ras oncogene (Rat-1-EJ) were analyzed for cell-associated <em>growth</em> <em>factor</em> activity. The two cell lines grew at the same rate, but at any given stage of <em>growth</em> the Rat-1-EJ cells synthesized two to four times more cell-associated <em>growth</em> <em>factor</em> activity than did the Rat-1 cells. For each cell line, the level of cell-associated <em>growth</em> <em>factor</em> activity was five to eight times greater at confluent densities compared to sparse densities. Heparin affinity chromatography and Western blot analysis demonstrated that the cell-associated <em>growth</em> <em>factor</em> was basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). The bFGF synthesized by the Rat-1-EJ cells appeared in two molecular mass forms, about 40% as an 18-kDa form which comigrated with recombinant bFGF and about 60% as a higher molecular mass doublet of about <em>22</em> kDa. The two forms of bFGF were biologically active and could be separated on a Mono S cation exchange column. Separation and purification to homogeneity of both the 18-kDa bFGF and the <em>22</em>-kDa bFGF doublet were achieved by a combination of CM-Sepharose cation exchange, heparin affinity-fast performance liquid chromatography, and C4 reverse phase high performance liquid chromatography.

A previous study showed that basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) promotes the effects of brain-derived neurotrophic <em>factor</em> (BDNF) on migration and neurite out<em>growth</em> from the cochleovestibular ganglion (CVG). This suggests that FGF-2 may up-regulate the receptor for BDNF. Thus we have examined TrkB expression during CVG formation and otic innervation in vitro and in the chicken embryo using immunohistochemistry. Following anatomical staging according to Hamburger-Hamilton, results were compared with mRNA expression in vitro using in situ hybridization. In the embryo at stage 16 (E2+) clusters of either lightly stained or immunonegative cells occurred within the otocyst and among those migrating to the CVG. By stage <em>22</em> (E3.5), immunostaining was concentrated in the CVG perikarya and invaded the processes <em>growing</em> into the otic epithelium but not into the rhombencephalon. Subsequently TrkB expression decreased in the perikarya and became localized in the leading processes of the fibers invading the epithelium and in the structures participating in synapse formation with the hair cells. In vitro there was moderate immunostaining and modest in situ hybridization for trkB in the neuroblasts migrating from the otocyst under control conditions. In contrast, neuroblasts previously exposed to FGF-2 exhibited accelerated migration and differentiation, with increased trkB mRNA expression. Morphological differentiation was associated with more intense immunostaining of processes than cell bodies. Evidently TrkB shifts its expression sequentially from sites engaged in migration, ganglion cell differentiation, axonal out<em>growth</em>, epithelial innervation, and synapse formation. FGF-2 may promote the role of BDNF in these developmental events by upregulating the TrkB receptor.

OBJECTIVE

To identify and critique the most recent experimental findings regarding the pathogenesis and therapy of thyroid-associated ophthalmopathy.

RESULTS

Much of the recent work in this field has focused on identifying genetic alterations associated with the phenotypes of Graves' disease and thyroid-associated ophthalmopathy and investigating their functional consequences. Identified candidate genes include CD40, cytotoxic T-lymphocyte antigen-4, protein tyrosine phosphatase-<em>22</em>, human leukocyte antigen-major histocompatibility complex and those associated with the X-chromosome. Efforts to generate a complete rodent model of Graves' disease continue with little progress. These uniformly involve the immunization of animals with the thyrotropin receptor. Studies conducted in vitro have focused on the actions of cytokines in orbital <em>fibroblasts</em>, the potential role of the insulin-like <em>growth</em> <em>factor</em>-1 receptor and activating antibodies directed against it as a <em>fibroblast</em> and T cell activation pathway. Reports continue to appear examining the potential relationship between the thyrotropin receptor and orbital adipogenesis. Regarding therapy for thyroid-associated ophthalmopathy, small molecules and antibodies disrupting cytokine pathways and lymphocyte function are currently under examination and have yielded promising albeit preliminary results.

CONCLUSIONS

Thyroid-associated ophthalmopathy remains a vexing medical problem, the pathogenesis of which remains uncertain. A number of obstacles continue to plague major advances, not least of which is the absence of a robust animal model. A few new insights seem to represent departure from traditional thinking about this disease and may herald important innovation.

1. The present study examined the role of C-phycocyanin (C-pc) in relation to <em>growth</em> <em>factors</em> and cell migration during wound healing. 2. Histological and biochemical studies showed that C-pc scaffold significantly (P < 0.01) increased hydroxyl proline, total hexamine and protein content, and decreased uronic acid content in the wound tissues during a time course study in newly formed skin. 3. Reverse transcription polymerase chain reaction array of mouse <em>growth</em> <em>factors</em> in wound tissue showed overexpression (up to 10-fold) of <em>growth</em> <em>factors</em>, such as Cxcl12, Fgf18, Lefty 1, Lefty 2, Rabep 1 and Zip91, and downregulation (up to -10-fold) of Amh, Bmp 7 and Nodal genes in a 6-day period in C-pc treated groups. Also, Csf 3, Fgf <em>22</em>, Mdk, Igf 2, transforming <em>growth</em> <em>factor</em> (TGF)-α 1 and interleukin (IL)-1β showed an upregulation of more than 30-fold than the control groups. TGF-β subfamily cytokine <em>growth</em> <em>factors</em>, such as Bmp 2, 4 and 8b, and other <em>growth</em> <em>factors</em>, such as Cxcl 1, showed the highest activity on day 3, showing a transient type of regulation. Western blot analysis showed a positive correlation between gene activity and protein expressions of Bmp 8b, Bmp4, Bmp2 and Cxcl 1. Day 6 in the C-pc group showed the highest csf3 and IL-1β expression. 4. C-pc had no direct effect on keratinocyte migration. However, keratinocytes that were co-cultured with <em>fibroblasts</em> showed a significantly higher rate of migration in the presence of C-pc, showing an indirect effect of C-pc on keratinocyte migration. 5. In conclusion, biodegradable C-pc scaffold might help to serve as an alternate scaffold material for wound healing.

Previous reports detailing mutational effects within the hydrophobic core of human acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-1) have shown that a symmetric primary structure constraint is compatible with a stably folded protein. In the present report, we investigate symmetrically related pairs of buried hydrophobic residues in FGF-1 (termed "mini-cores") that are not part of the central core. The effect upon the stability and function of FGF-1 mutations designed to increase primary structure symmetry within these "mini-core" regions was evaluated. At symmetry-related positions <em>22</em>, 64, and 108, the wild-type protein contains either Tyr or Phe side chains. The results show that either residue can be readily accommodated at these positions. At symmetry-related positions 42, 83, and 130, the wild-type protein contains either Cys or Ile side chains. While positions 42 and 130 can readily accommodate either Cys or Ile side chains, position 83 is substantially destabilized by substitution by Ile. Tertiary structure asymmetry in the vicinity of position 83 appears responsible for the inability to accommodate an Ile side chain at this position, and is known to contribute to functional half-life. A mutant form of FGF-1 with enforced primary structure symmetry at positions <em>22</em>, 64, and 108 (all Tyr) and 42, 83, and 130 (all Cys) is shown to be more stable than the reference FGF-1 protein. The results support the hypothesis that a symmetric primary structure within a symmetric protein superfold represents a solution to achieving a foldable, stable polypeptide, and highlight the role that function may play in the evolution of asymmetry within symmetric superfolds.

Using a sterol auxotroph of the LM cell mouse <em>fibroblast</em>, we demonstrate that relatively few cholesterol analogues can substitute for cholesterol as a <em>growth</em> <em>factor</em>. The auxotroph grows normally on desmosterol and trans-<em>22</em>-dehydrocholesterol and at reduced rates on dihydrocholesterol, campesterol, and <em>22</em>,23-dihydrobrassicasterol. It does not grow with beta-sitosterol, stigmasterol, ergosterol, or cis-<em>22</em>-dehydrocholesterol when the sterol is present as sole supplement but does grow at normal rates when the analogue is supplied with suboptimal amounts of cholesterol. Two contrasting types of membrane lipid changes are observed in cells grown on cholesterol analogues. In cells grown with dihydrocholesterol, a marked increase in desaturation and elongation of fatty acids is noted. Conversely, when cells are grown with cis-<em>22</em>-dehydrocholesterol, desaturation and elongation of fatty acids are severely curtailed. Cells grown on alkyl sterols respond like cells grown on cis-<em>22</em>-dehydrocholesterol but in a less pronounced fashion. The effects of sterol substitution in mammalian cells versus in lower eukaryotes are compared, and an explanation for the secondary changes in fatty acid composition in terms of phospholipid phase behavior is suggested.

<em>Fibroblasts</em> in culture and leukocytes have been widely used to study fatty acid and lipoprotein cellular metabolism. The present investigations were designed to study the role of nutritional and environmental <em>factors</em> on lipid metabolism in these two types of cells. Leukocytes freshly isolated from human blood and <em>fibroblasts</em> cultured in media enriched in human serum (HS) have relatively similar fatty acid distributions. However, more important differences are observed in <em>fibroblasts</em> cultured in media enriched with HS or with fetal bovine serum (FBS). It is obvious that the quantity and quality of fatty acids are very different in FBS and HS, but intracellular regulation ensures relative homogeneity of saturated (SFA) and monounsaturated fatty acids (MUFA) in the cells, particularly in phospholipids. The first modifications induced by different media (FBS or HS) are detected on cellular <em>growth</em>; the differences seem to be due more to the fatty acid (FA) quantitative supply than to the FA quality of each culture medium. The major modifications in FA composition induced by different culture media concern the polyunsaturated fatty acids (PUFA) of phospholipids, especially the n-6 family. The intracellular linoleic acid level depends on the level in the medium, but intracellular n-6 metabolite levels depend both on the level in the medium and on the <em>growth</em> state of the cells. The n-3 family seems to be less affected by the quality of the medium in our experiment, and the cells maintain a stable docosahexaenoic acid (<em>22</em>:6n-3) level. A higher content of the n-3 family in the medium induces a higher level of eicosa- or docosapentaenoic acid, rather than docosahexaenoic acid itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadotropin-induced ovulation is associated with oocyte maturation and preovulatory increases of tissue plasminogen activator (tPA) expression. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), an angiogenic <em>factor</em> found in many organs including the ovary, modulates steroidogenesis in granulosa cells and increases PA activity in endothelial cells. Here studies were performed to examine the possible roles of bFGF as an intragonadal regulator of tPA expression and oocyte maturation. In cultured granulosa cells, bFGF caused a time-dependent (onset at 24 h) and dose-dependent (ED50 = 0.6 nM) increase (up to 5-fold) in tPA enzyme activity as measured by the fibrin overlay technique. Northern blot hybridization also revealed that treatment of cells with bFGF (2 nM) increased the level of the <em>22S</em> tPA messenger RNA. Slot blot analysis indicated that the effects of bFGF were time dependent and dose dependent; tPA message levels increase before tPA activity levels. bFGF (0.6 nM) also significantly increased granulosa cell cAMP production in both the absence and presence of a phosphodiesterase inhibitor. In follicle-enclosed oocytes incubated for 24 h in media with or without increasing concentrations of LH or bFGF, germinal vesicle breakdown was observed in only 1.6% of controls, but 85% of LH (1 microgram/ml)-treated oocytes underwent maturation. Likewise, bFGF induced germinal vesicle breakdown (10-80%) over a dose range of 0.6 to 333 nM. In the same follicles, bFGF, like LH, also stimulated prostaglandin E production. These results, coupled with the identification of bFGF in <em>growing</em> follicles, suggest that bFGF acts as an intraovarian inducer of granulosa cell tPA gene expression and oocyte maturation.

BACKGROUND

Connective tissue growth factor (CTGF/CCN2), a multifunctional protein that regulates cell growth and differentiation, is known to play an important role in tumourigenesis of several human malignancies. However, CCN2 expression or its potential role in head and neck squamous cell carcinoma (HNSCC) is not known, even though HNSCC is one of the most common cancers worldwide.

OBJECTIVE

To investigate CCN2 expression in primary HNSCC and to correlate CCN2 mRNA expression level with one of its upstream regulators, transforming growth factor-beta1 (TGF-beta1).

METHODS

Tissue specimens of HNSCC (n = 22) and normal oral mucosa (n = 8) were analysed by real-time, quantitative PCR assays for CCN2 and TGF-beta1 expression. Tissue localisation of CCN2 protein was analysed by immunohistochemistry.

RESULTS

Primary HNSCC expressed high levels of CCN2 mRNA. CCN2 protein was localised in stromal fibroblasts, tumour and vascular endothelial cells.

CONCLUSIONS

Results show that CCN2 mRNA and protein are overexpressed in HNSCC, suggesting that CCN2 expression should be further evaluated for a possible role in HNSCC growth and progression.

OBJECTIVE

The role of reversibility of nontraditional risk factors, like inflammation and CKD-mineral bone disorder, in the reduction of cardiovascular risk after renal transplantation is still scarcely defined.

METHODS

The longitudinal relationship between C-reactive protein, CKD-mineral bone disorder biomarkers, and intima media thickness was investigated in a series of 178 patients (age=32±10 years) with stage 5 CKD maintained on chronic dialysis who underwent echo-color Doppler studies of the carotid arteries before and after renal transplantation. Smokers and patients with diabetes were excluded from the study. In all patients, immunosuppression was performed by a standard regimen on the basis of calcineurin inhibitors. Healthy controls were specifically selected to match the age and sex distribution of the patients. Biochemical and intima media thickness assessments were repeated 6 months after transplantation.

RESULTS

Before transplantation, intima media thickness in patients with stage 5 CKD on dialysis (average=0.9±0.2 mm) was higher (P<0.001) than in well matched healthy controls (0.6±0.1 mm) and reduced substantially (-22%; 95% confidence interval, -24% to -20%) after transplantation (P=0.001). GFR (multivariable-adjusted β=0.23; P<0.001), C-reactive protein (β=0.15; P<0.001), and fibroblast growth factor 23 (β=0.28; P<0.001) were the strongest independent correlates of intima media thickness before transplantation. Similarly, longitudinal changes in the same biomarkers were the sole independent correlates of simultaneous changes in intima media thickness (C-reactive protein: β=0.25; fibroblast growth factor 23: β=0.26; P<0.001 for both) after renal transplantation. The evolution of intima media thickness after transplantation was largely independent of classic risk factors, including BP, LDL cholesterol, and insulin resistance, as measured by homeostatic model assessment.

CONCLUSIONS

Intima media thickness improves after renal transplantation. Such an improvement associates with parallel changes in serum C-reactive protein and fibroblast growth factor 23. These observations are in keeping with the hypothesis that the decline in cardiovascular risk after transplantation, in part, depends on partial resolution of nontraditional cardiovascular risk factors, like inflammation and CKD-mineral bone disorder.

Gene expression of <em>fibroblast</em> <em>growth</em> <em>factor</em>-9 (FGF9) is decreased in granulosa cells (GC) of cystic follicles compared with normal dominant follicles in cattle. The objectives of this study were to investigate the effects of FGF9 on GC steroidogenesis, gene expression, and cell proliferation and to determine the hormonal control of GC FGF9 production. GC were collected from small (1-5 mm) and large (8-<em>22</em> mm) bovine follicles and treated in vitro with various hormones in serum-free medium for 24 or 48 h. In small- and large-follicle GC, FGF9 inhibited (P < 0.05) IGF-I-, dibutyryl cAMP-, and forskolin-induced progesterone and estradiol production. In contrast, FGF9 increased (P < 0.05) GC numbers induced by IGF-I and 10% fetal calf serum. FGF9 inhibited (P < 0.05) FSHR and CYP11A1 mRNA abundance in small- and large-follicle GC but had no effect (P>> 0.10) on CYP19A1 or StAR mRNA. In the presence of a 3β-hydroxysteroid dehydrogenase inhibitor, trilostane, FGF9 also decreased (P < 0.05) pregnenolone production. IGF-I inhibited (P < 0.05) whereas estradiol and FSH had no effect (P>> 0.10) on FGF9 mRNA abundance. TNFα and wingless-type mouse mammary tumor virus integration site family member-3A decreased (P < 0.05) whereas T(4) and sonic hedgehog increased (P < 0.05) FGF9 mRNA abundance in control and IGF-I-treated GC. Thus, GC FGF9 gene expression is hormonally regulated, and FGF9 may act as an autocrine regulator of ovarian function by slowing follicular differentiation via inhibiting IGF-I action, gonadotropin receptors, the cAMP signaling cascade, and steroid synthesis while stimulating GC proliferation in cattle.

BACKGROUND

The proliferative response of type II cells is an important event following silica-induced lung injury. Alveolar macrophages, when activated by fibrogenic agents, secrete various biological mediators which affect cell growth.

METHODS

Human alveolar macrophages from normal volunteers were incubated in serum-free medium or in the presence of increasing concentrations of silica. Alveolar macrophage conditioned media were diluted and added to type II cell cultures for proliferation studies. Purified type II pneumocytes were isolated from fetal rat lungs for bioassays. Growth factor activities were partially characterised by size exclusion chromatography. Each fractionated mitogenic peak was preincubated with monoclonal antibody against platelet derived growth factor (PDGF) or antisera against insulin-like growth factor 1 (IGF-1) or fibroblast derived growth factor (FGF) in order to study the nature of each activity.

RESULTS

Conditioned media from alveolar macrophages exposed to silica induced an increase in type II cell DNA synthesis and cell number over that seen when type II cells were incubated with unstimulated alveolar macrophage supernatants. Size exclusion of alveolar macrophage supernatants exposed to silica showed four peaks of type II cell stimulating activity with apparent molecular weights of 38, 22, 16, and 8 kDa. Anti-PDGF antibody significantly reduced the activity of the first and second peaks, antiserum against IGF-1 partially reduced the activity of the first and fourth peaks, and antiserum against FGF reduced only the third peak of activity.

CONCLUSIONS

Human alveolar macrophages exposed to silica in vitro release mitogens for type II pneumocytes including PDGF-like, IGF-1-like, and FGF-like molecules. These agents are likely to be involved in the epithelial repair and type II cell hyperplasia observed in silicosis.

Runx2 and Sp7 transcription <em>factors</em> are essential for skeletogenesis. Targeted deletion of either gene results in failure of osteoblast differentiation and bone formation. Loss of bone-matrix gene expression is surprisingly similar in Sp7 and Runx2 null mice. The molecular mechanisms responsible for similar transcriptional regulation of target genes remain largely unknown. Here, we demonstrate that Runx2 and Sp7 interact physically and functionally. Both proteins are co-expressed in osteoblastic cells. We first characterized a panel of Sp7 antibodies and demonstrate that majority of the published antibodies do not recognize Sp7 protein. Co-immunoprecipitation studies revealed that endogenous Runx2 protein physically interacts with Sp7 protein. We identified that runt homology domain (RHD) of Runx2 protein is involved in physical association with Sp7. Functional consequences of Runx2-Sp7 physical interaction was then assessed by promoter-reporter assays. We selected promoters of osteocalcin (OC), a marker of mature osteoblast and <em>fibroblast</em> <em>growth</em> <em>factor</em> 3 (FGF3), a signaling molecule that determine the fate of embryonic ecto-mesenchyme. Runx2 and Sp7 stimulate OC-promoter activity by 3-folds in epithelial cells. However, when both proteins were co-expressed, a dose-dependent synergistic activation of <em>22</em>-folds was noted. Similar pattern of synergistic activation of OC-promoter was noted in mesenchymal cell. FGF3 promoter was activated by 25 - and 30-folds with Runx2 and Sp7 respectively. Again a dose-dependent synergistic activation of 130-folds was evident when Runx2 and Sp7 were co-expressed in epithelial cells. Synergistic activation of FGF3 promoter was also noted in mesenchymal cells. Together, our data demonstrated that Runx2-Sp7 molecular complex functionally cooperate for maximal induction of cell-phenotype-restricted genes.

Filamins regulate cell locomotion and associate with diverse signaling molecules. We have recently found that targeting filamin A (FLNA) reduces RAS-induced lung adenocarcinomas. In this study, we explored the role of another major filamin isoform, filamin B (FLNB), in tumor development. In contrast to FLNA, we report that targeting FLNB enhances RAS-induced tumor <em>growth</em> and metastasis which is associated with higher matrix metallopeptidase-9 (MMP-9) and extracellular signal-regulated kinase (ERK) activity. Flnb deficiency in mouse embryonic <em>fibroblasts</em> results in increased proteolytic activity of MMP-9 and cell invasion mediated by the RAS/ERK pathway. Similarly, silencing FLNB in multiple human cancer cells increases the proteolytic activity of MMP-9 and tumor cell invasion. Furthermore, we observed that Flnb-deficient RAS-induced tumors display more capillary structures that is correlated with increased vascular endothelial <em>growth</em> <em>factor</em>-A (VEGF-A) secretion. Inhibition of ERK activation blocks phorbol myristate acetate-induced MMP-9 activity and VEGF-A secretion in vitro. In addition, silencing FLNB in human ovarian cancer cells increases secretion of VEGF-A that induces endothelial cells to form more vascular structures in vitro. We conclude that FLNB suppresses tumor <em>growth</em> and metastasis by regulating the activity of MMP-9 and secretion of VEGF-A which is mediated by the RAS/ERK pathway.Oncogenesis (2014) 3, e119; doi:10.1038/oncsis.2014.33; published online <em>22</em> September 2014.

OBJECTIVE

To observe the clinical efficacy of Chinese medicine (CM) treatment of Hongyou Ointment and Shengji Powder on diabetic ulcers, and to observe the influence of CM treatment on the expressions of proteins associated with the Wnt signaling pathway, such as β-catenin, c-myc and K6.

METHODS

sixty-two patients fitting the registration standards were randomly divided into the CM group (31 patients) and the Western medicine (WM) group (31 patients) by a random number table. The patients in the CM group were treated with Hongyou Ointment and Shengji Powder externally. The patients in the WM group were treated with mupirocin ointment, growth factor (bFGF), and Vaseline gauze for external use and with basic therapies. Wound-healing time and four-week healing rate were recorded. The wounds were measured by digital photography and ImageJ software. Skin biopsies were obtained from 24 patients before CM treatment and 20 patients after CM treatment. Immunohistochemical tests and semi-quantitative imaging with NIH ImageJ 1.42 software were used to analyze the changes in protein expression of β-catenin, c-myc, and K6.

RESULTS

Fifty-three patients completed the trial; four patients in the CM treatment group and five patients in the WM group dropped out. Among them, four were dissatisfied with the treatment process, two could not continue because of their jobs, two failed to complete the course of follow-up appointments, and one was diagnosed with squamous cell carcinoma during treatment. The comparison of ulcer healing rates between the two groups showed insignificant differences (P=0.77). The ulcer healing rates were 33.33% (9/27) in the CM group and 26.92% (7/26) in the WM group. However, the effective rate was significantly higher in the CM group (81.48%, 22/27) than in the WM group (57.69%, 15/26, P=0.04). The mean wound healing time was shorter in the CM group (22.71 ±5.46 days) than in the WM group (26.56 ±7.56 days, P=0.04). CM treatment was well tolerated, and there was no withdrawal due to adverse reactions. Immunohistochemical analysis in the refractory wound indicated higher expressions of β-catenin, c-myc and K6 compared with the normal skin. β-catenin was abnormally expressed in the nuclei of the keratinocytes and fibroblasts at the wound margins, and the expressions of c-myc and K6 were highly expressed in the full hyperplastic epidermis, especially in the granular layer (P<0.05). The expressions of these proteins decreased after CM treatment. The expression levels of β-catenin, c-myc, and K6 proteins before and after the treatment were 101.88 ± 10.76 vs. 140.42 ±8.45; 113.27 ± 16.75 vs. 153.79 ±8.32; 90.39 ±11.07 vs. 151.29 ±7.39, respectively.

CONCLUSIONS

CM treatment using Hongyou Ointment and Shengji Powder was efficient in the management of diabetic skin ulcers. The mechanism of action might be related to the inhibition of the Wnt signaling pathway.

There is no standard or known optimal treatment for idiopathic pulmonary fibrosis. Corticosteroids have been used with widely variable benefit. Colchicine has been reported to suppress <em>fibroblast</em> <em>growth</em> <em>factors</em> and to inhibit collagen deposition. A potential role has been proposed for colchicine in the treatment of fibrotic pulmonary diseases. We retrospectively assessed the outcome of 23 patients with idiopathic pulmonary fibrosis in whom colchicine was used as empiric treatment. Eighteen patients had received prior corticosteroid therapy. By clinical and pulmonary function criteria, five patients (<em>22</em> percent) improved following colchicine, nine (39 percent) remained stable, and nine (39 percent) worsened. The average duration of follow-up was <em>22</em> months. Despite limitations of this retrospective analysis, colchicine may be of benefit in pulmonary fibrosis and should be considered for further clinical trials.