Differential expression of TGF-beta isoforms during differentiation of HaCaT human keratinocyte cells: implication for the separate role in epidermal differentiation

The three mammalian isoforms of transforming growth factor-beta(TGF-beta1, beta2, beta3) are potent regulators of cell growth, differentiation, and extracellular matrix deposition. To study their role in skin differentiation, we investigated the expression of TGF-beta isoforms on cell growth and differentiation induction of the human keratinocyte cell line, HaCaT by elevating the Ca(2+) concentration. An ELISA and RT-PCR assay revealed secreted TGF-beta 1 protein and TGF-beta 1 mRNA were increased during calcium-induced differentiation. In contrast, major differences were seen for TGF-beta 2 and TGF-beta 3 mRNA which were decreased during differentiation, but TGF-beta 2 and TGF-beta3 protein were not evident on an ELISA. These results suggest different functions for each TGF-beta isoforms in epidermal differentiation, such that TGF-beta 1 is associated with the more differentiated state, and TGF-beta 2 and TGF-beta 3 may be associated the more proliferated state.     

Insulin-like Growth Factors Promote DNA Synthesis and Support Cell Viability in Fetal Hemopoietic Tissue by Paracrine Mechanisms

Abstract

There is significant evidence that the insulin-like growth factors (IGF) play a role in both murine and human hemopoiesis. In order to better define the nature and mechanisms of these effects, we have used a serum-free system to examine DNA synthesis and cell replication in murine hemopoietic cells. Cell preparations from 13-day fetal mice livers were incubated in serum-free DMEM alone or with erythropoetin (Epo) 0.5 U/ml, recombinant human IGF-I, purified IGF-II, or recombinant human growth hormone (GH) in various doses, and [³H]thymidine added for the last 3 hr of 21-hr incubation. Cell distribution was over 80% erythroid or erythroblasts. IGF-I and IGF-II promoted thymidine incorporation into cells at a half-maximal dose of 3 and 1 nM respectively, IGF-II with a maximum potency 65% of IGF-I; insulin stimulated at a half-maximum dose of 100 nM, with similar maximum effect to IGF-I, and their effects were not additive. GH was stimulatory at 1 μM. Epo was 2–9 times as effective as IGF-I and their effects were not additive. A monoclonal antibody to IGF-I reduced the effect of IGF-I by 50–80%, had no effect on Epo, and abolished the GH effect. Separation of erythroid cells and precursors from accessory and other liver cells did not alter the response to IGF-I. Cell counts increased in response to IGF-I or Epo, and cell viability was maintained by IGF-I compared to control medium.

We demonstrate that (1) physiologic levels of IGF-I and IGF-II promote DNA synthesis in these cells, (2) their effects are primarily on erythroid precursors, (3) insulin's effects are probably via the IGF-I receptor, (4) the effects of IGF-I are specific and direct, and primarily not generated by a product of accessory cells, (5) high-dose GH has effects which are probably via local IGF production. It is concluded that IGF plays a role in fetal hemopoiesis, both by promoting cell growth and by maintenance of viability.     

Epithelial-Mesenchymal Transitions of Bile Duct Epithelial Cells in Primary Hepatolithiasis

The purpose of this study was to explore the role of epithelial-mesenchymal transition in the pathogenesis of hepatolithiasis. Thirty-one patients with primary hepatolithiasis were enrolled in this study. Expressions of E-cadherin, α-catenin, α-SMA, vimentin, S100A4, TGF-β1 and P-smad2/3 in hepatolithiasis bile duct epithelial cells were examined by immunohistochemistry staining. The results showed that the expressions of the epithelial markers E-cadherin and α-catenin were frequently lost in hepatolithiasis (32.3% and 25.9% of cases, respectively), while the mesenchymal markers vimentin, α-SMA and S100A4 were found to be present in hepatolithiasis (35.5%, 29.0%, and 32.3% of cases, respectively). The increased mesenchymal marker expression was correlated with decreased epithelial marker expression. The expressions of TGF-β1 and P-smad2/3 in hepatolithiasis were correlated with the expression of S100A4. These data indicate that TGF-β1-mediated epithelial-mesenchymal transition might be involved in the formation of hepatolithiasis.     

Epidermal Growth Factor Receptor Mutations and the Clinical Outcome in Male Smokers with Squamous Cell Carcinoma of Lung

Epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) have been reported to be related to certain clinical characteristics (i.e., female, non-smokers with adenocarcinoma) and gefitinib responsiveness. This exploratory analysis was performed to determine the incidence of EGFR mutations in male smokers with squamous cell carcinoma, who were treated with EGFR tyrosine kinase inhibitor, gefitinib. Sixty-nine Korean NSCLC patients were treated with gefitinib in a prospective study. For a subset of 20 male patients with squamous cell carcinoma and a history of smoking, pretreatment tumor tissue samples were obtained and analyzed for EGFR mutations (exons 18 to 21). EGFR mutations were found in 3 (15%) patients, including in-frame deletions within exon 19 (n=2) and L858R missence mutation in exon 21 (n=1). These 3 patients with EGFR mutations responded to gefitinib, whereas only one of remaining 17 patients with wild-type EGFR achieved clinical response. Trend toward longer progression-free (5.8 vs. 2.4 months; P=0.07) was noted in patients with EGFR mutations compared to those with wild-type EGFR. Although male smokers with squamous cell carcinoma have not been considered ideal candidates for gefitinib treatment, significant incidence of EGFR mutations was observed. The molecular markers should be considered to predict clinical benefits from gefitinib.     

Insulin-like Growth Factor 1 (IGF-1) as a marker of cognitive decline in normal ageing: A review

Insulin-like Growth Factor 1 (IGF-1) and its signaling pathway play a primary role in normal growth and ageing, however serum IGF-1 is known to reduce with advancing age. Recent findings suggest IGF-1 is essential for neurogenesis in the adult brain, and this reduction of IGF-1 with ageing may contribute to age-related cognitive decline. Experimental studies have shown manipulation of the GH/GF-1 axis can slow rates of cognitive decline in animals, making IGF-1 a potential biomarker of cognition, and/or its signaling pathway a possible therapeutic target to prevent or slow age-related cognitive decline. A systematic literature review and qualitative narrative summary of current evidence for IGF-1 as a biomarker of cognitive decline in the ageing brain was undertaken. Results indicate IGF-1 concentrations do not confer additional diagnostic information for those with cognitive decline, and routine clinical measurement of IGF-1 is not currently justified. In cases of established cognitive impairment, it remains unclear whether increasing circulating or brain IGF-1 may reverse or slow down the rate of further decline. Advances in neuroimaging, genetics, neuroscience and the availability of large well characterized biobanks will facilitate research exploring the role of IGF-1 in both normal ageing and age-related cognitive decline.     

Effect of Pro-inflammatory/Anti-inflammatory Agents on Cytokine Secretion by Peripheral Blood Mononuclear Cells in Rheumatoid Arthritis and Systemic Lupus Erythematosus

We studied a well-selected population of patients with active rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) without immunosuppressive therapy. Control and patient peripheral blood mononuclear cells (PBMC) were incubated with IL-1 &#103, IL-10, TGF- &#103 or LPS for 20 h and the in vitro basal and stimulated secretions of IL-6, TNF- &#102, IL-1 &#103 and IL-1ra were measured by ELISA. We found that in the SLE patients the basal secretion of IL-6 was significantly lower and that of IL-1ra significantly higher than in control subjects, while in the RA group the basal IL-1ra secretion was higher than in healthy subjects. SLE and RA PBMC responded to LPS and IL-1 &#103 reaching higher cytokine secretion values than controls. The in vitro response of SLE and RA PBMC to TGF &#103 was normal, while that to IL-10 was defective: IL-10 was able to stimulate the production of IL-6 and IL-1ra in PBMC from normal subjects, but it was unable to enhance IL-6 secretion in RA cells and it was also completely ineffective in inducing IL-1ra secretion in both SLE and RA PBMC. Our work add new data useful for the evaluation of IL-10 and IL-1ra as therapeutic agents in rheumatic diseases.     

Expression of Insulin-like Growth Factors (IGF)-1 and -2, IGF-binding Proteins-2 and -3, and Receptors for Growth Hormone,IGF Type-1 and -2 and Insulin in the Gastrointestinal Tract of Neonatal Calves

Growth hormone (GH), insulin-like growth factors (IGFs) and insulin influence post-natal gastrointestinal development and function. We have measured by real-time PCR the mRNA levels of IGF-1 and -2, IGF-binding proteins (IGFBPs)-2 and -3, and receptors for GH, IGF type-1 and -2, and insulin in esophagus, rumen, fundus, pylorus, duodenum, jejunum, ileum and colon of calves on days 1 and 5 of life. Levels of mRNA of measured traits were different (P<0.05) at different gastrointestinal sites. Furthermore, mRNA levels of IGFs, IGFBPs and of receptors for GH and IGF type-1 and -2 and insulin differed (P<0.05) on days 1 and 5. Differences in mRNA abundance of IGFs, IGFBPs and of receptors for GH, IGFs, and insulin among gastrointestinal sites on days 1 and 5 of life suggest site-specific functional importance and demonstrate that changes are the consequence of ontogenetic development and/or due to feeding.     

Effects of All-Trans-Retinoic Acid (atRA) on Inducible Nitric Oxide Synthase (iNOS) Activity and Transforming Growth Factor Beta-1 Production in Experimental Anti-GBM Antibody-Mediated Glomerulonephritis

Sustained high output release of Nitric oxide (NO) as result of activation of inducible nitric oxide synthase (iNOS), and increased production of the antiproliferative/profibrotic cytokine transforming growth factor-1 (TGF-1) are well documented in glomerulonephritis. Modulation of iNOS activity and of TGF-1 production can therefore be viewed as anti-inflammatory strategies. The present study employed all-transretinoic acid (atRA) which is known to have anti-inflammatory effects and to modulate expression of iNOS and TGF-1, in order to explore its effect on iNOS enzyme activity and TGF-1 production in anti-GBM antibody induced glomerulonephritis. Glomerulonephritis was induced in Lewis rats by injection of anti-GBM antibody. A group of nephritic rats were given daily administration of atRA for 14–16 days. Extent of proteinuria was assessed by measuring urine protein and creatinine excretion. iNOS enzyme activity was measured by calculating conversion of L[¹⁴C]arginine to L-[¹⁴C]citrulline in glomerular protein lysates. Levels of TGF-1 in glomerular protein lysates were measured by quantitative ELISA. Levels of proliferating nuclear antigen (PCNA), TGF- receptor II (TGF-RII), and fibronectin were assessed by Western blot analysis. Glomerular iNOS activity in atRA treated nephritic animals was attenuated in comparison to that in nephritic controls that were not. Glomerular expression of PCNA was also reduced. Levels of TGF-1 were increased in glomeruli of atRA treated nephritic animals. In these animals, there was no change in glomerular levels of TGF- receptor II (TGF-RII) or fibronectin, and there was no reduction in urine protein excretion. These results suggest that atRA attenuates iNOS activity and proliferation in glomeruli of nephritic animals. The failure of atRA treatment to reduce proteinuria could be due to the increase in TGF-1 levels and to inhibition of iNOS-driven NO production.     

Challenge to Predict Mobilized Peripheral Blood Stem Cells on the Fourth Day of Granulocyte Colony-Stimulating Factor Treatment in Healthy Donors: Predictive Value of Basal CD34+ Cell and Platelet Counts

A longitudinal, prospective, observational, single-center cohort study on healthy donors was designed to identify predictors of CD34⁺ cell mobilization on day 4 after granulocyte colony-stimulating factor (G-CSF) administration. As potential predictors of mobilization, age, sex, body weight, height, blood volume, WBC count, peripheral blood (PB) mononuclear cell count, platelet (Plt) count, and hematocrit and hemoglobin levels were considered. Two different evaluations of CD34⁺ cell counts were determined for each donor: baseline (before G-CSF administration) and in PB on day 4 after G-CSF administration. One hundred twenty-two consecutive healthy donors with a median age of 47.5 years were enrolled. The median value of CD34⁺ on day 4 was 43 cells/µL (interquartile range, 23 to 68), and 81.1% of donors had ≥20 cells/µL. Basal WBC count, Plt count, and CD34⁺ were significantly higher for the subjects with CD34⁺ levels over median values on day 4. A multivariate quartile regression analysis, adjusted by sex, age, basal CD34⁺, and basal Plt count, showed a progressively stronger relationship between baseline CD34⁺ and Plt levels and the CD34⁺ levels on day 4. The basal CD34⁺ cut-off level to predict the levels of CD34⁺ on day 4 was either ≤2 cells/μL or ≥3 cells/μL and that of basal Plt count was ≤229 × 10⁹/L or ≥230 × 10⁹/L, respectively, to determine whether mobilization therapy should or should not be attempted. PB stem cell mobilization with G-CSF was highly effective on day 4, and herein we describe a model for predicting the probability of performing PB stem cell collection after a short course of G-CSF.     

Regulation of Interleukin (IL)-6 and IL-8 Production in an Amnion-derived Cell Line by Cytokines,Growth Factors,Glucocorticoids, and Phorbol Esters

PROBLEM: To determine whether amnion cells produce interleukin (IL)?6 and ?8 and thus may contribute to the high concentrations of these cytokines in amniotic fluid at term. METHOD OF STUDY: Amnion-derived WISH cells were treated in culture with stimuli over 16 hr, and IL-6 and IL-8 concentrations in the conditioned media were measured by enzyme-linked immunosorbent assay or bioassay (IL-6 only). RESULTS: IL-8 production was ?5-fold higher than that of IL-6 under basal and stimulated conditions. Significant (by Dunnett's test after analysis of variance) stimulation of production of both cytokines was achieved by IL-1β (>0.2 ng/ml), TNFα (>10 ng/ml), and the phorbol ester, phorbol 12-myristate 13-acetate (>2 nM), over a 16-hr culture period. Epidermal growth factor at 10 ng/ml induced a small increase in production of IL-8, but not of IL-6, whereas bacterial lipopolysaccharide had minimal effects on production of either cytokine. Basal and cytokine-stimulated IL-6 and IL-8 production was inhibited by dexamethasone at concentrations equal to or greater than 1 nM. CONCLUSION: These findings suggest that amnion may be a significant contributor to the IL-6 and IL-8 content of amniotic fluid, and that WISH cells may be a suitable model for the study of cytokine production by amnion epithelial cells.