Smad3 Gene Expression Research Articles

Preconditioning Human Mesenchymal Stem Cells with a Low Concentration of BMP2 Stimulates Proliferation and Osteogenic Differentiation In Vitro.

Clinical trials using bone morphogenetic protein-2 (BMP2) for bone reconstruction have shown promising results. However, the relatively high concentration needed to be effective raises concerns for efficacy and safety. The aim of this study was to investigate the osteogenic effect of an alternative treatment strategy in which human bone marrow–derived mesenchymal stem cells (hMSCs) are preconditioned with low concentrations of BMP2 for a short time in vitro. hMSCs in suspension were stimulated for 15 min with 10 and 20 ng/mL of BMP2. After the BMP2 was removed, the cells were seeded and cultured in osteogenic medium. The effects of preconditioning were analyzed with regard to proliferation and expression of osteogenic markers at both gene and protein level. The results were compared to those from cultures with continuous BMP2 stimulation. A significant increase in proliferation was seen with both precondition and continuous stimulation with BMP2, with no difference between the treatments. Preconditioning with BMP2 significantly increased gene expression of RUNX2, COLI, ALP, and OC, and protein levels of COLI and ALP. This was not found with continuous stimulation. The role of preconditioning with BMP2 in osteogenesis was validated by findings of increased gene expression of SMAD1 and an increase in dual phosphorylation of ser 463 and ser 465 in the SMAD 1/5/8 pathway. We concluded that preconditioning hMSCs with BMP2 stimulates osteogenesis: proliferation with matrix secretion and matrix maturation of hMSCs. This implies that preconditioning with BMP2 might be more effective at inducing proliferation and osteogenic differentiation of hMSCs than continuous stimulation. Preconditioning with BMP2 could benefit the clinical application of BMP2 since side effects from high-dose treatments could be avoided.

TIMP-1 expression in human colorectal cancer is associated with SMAD3 gene expression levels: a pilot study.

The prognosis of colorectal cancer (CRC) varies considerably, and there is a compelling need to identify novel biomarkers with prognostic significance. The aim of the present study was to evaluate the prognostic value of a panel of six genes (CDH1, SMAD3, TGFβ1, ICAM-1, TIMP-1 and MUC12) in CRC patients. METHODS. We evaluated these genes by qRT-PCR in normal and CRC tumor tissue, and correlated the relative gene expression values with clinical, pathological aspects and other biological factors. RESULTS. RNA expression levels of CDH1, SMAD3, TGFβ1, ICAM-1, TIMP-1 and MUC12 were measured by qRT-PCR in a set of 39 tumor samples and non-cancer tissue. Statistically significant increases in expression levels were found for ICAM-1 and TIMP-1 when comparing tumor samples to the non-tumor group. CONCLUSIONS. Among the genes which displayed differential expressions between tumor tissue and adjoining normal tissue, the ones that presented statistically significant correlations were TIMP-1 and SMAD3, possibly with prognostic significance.

Effects of concurrent strength and endurance training on genes related to myostatin signaling pathway and muscle fiber responses.

Concurrent training (CT) seems to impair training-induced muscle hypertrophy. This study compared the effects of CT, strength training (ST) and interval training (IT) on the muscle fiber cross-sectional area (CSA) response, and on the expression of selected genes involved in the myostatin (MSTN) signaling mRNA levels. Thirty-seven physically active men were randomly divided into 4 groups: CT (n = 11), ST (n = 11), IT (n = 8), and control group (C) (n = 7) and underwent an 8-week training period. Vastus lateralis biopsy muscle samples were obtained at baseline and 48 hours after the last training session. Muscle fiber CSA, selected genes expression, and maximum dynamic ST (1 repetition maximum) were evaluated before and after training. Type IIa and type I muscle fiber CSA increased from pre- to posttest only in the ST group (17.08 and 17.9%, respectively). The SMAD-7 gene expression significantly increased at the posttest in the ST (53.9%) and CT groups (39.3%). The MSTN and its regulatory genes ActIIb, FLST-3, FOXO-3a, and GASP-1 mRNA levels remained unchanged across time and groups. One repetition maximum increased from pre- to posttest in both the ST and CT groups (ST = 18.5%; CT = 17.6%). Our findings are suggestive that MSTN and their regulatory genes at transcript level cannot differentiate muscle fiber CSA responses between CT and ST regimens in humans.

Alcohol accentuates simian immunodeficiency virus‐induced alterations in the skeletal muscle milieu contributing to impaired satellite cell myogenic differentiation (732.5)

Muscle wasting adversely affects survival in people living with HIV/AIDS (PLWHA). Studies using a non‐human primate model of SIV infection have demonstrated that chronic binge alcohol (CBA) consumption accentuates muscle wasting in AIDS. Alcohol‐accentuation of muscle wasting appears to be due to increased protein degradation and inability to rebuild muscle mass, which we predict is due to an inability of satellite cells (SCs), the precursors of skeletal muscle fibers, to form new myofibers. We have previously demonstrated that CBA impairs the ex vivo myogenic differentiation of SCs, the muscle progenitor cells. Moreover, there was a significant increase in Smad2 gene expression, the downstream intermediate of transforming growth factor‐ß1 (TGF‐B1) signaling, in CBA SCs suggesting a role of this pathway in inhibiting myogenesis. Based on our preliminary studies and the suggestion that TGF‐ß1 may negatively regulate SC activation, we hypothesized that the chronic inflammatory and oxidative skeletal muscle milieu leads to a profibrotic phenotype of SCs due to activation of TGF‐β1 inhibiting their myogenic differentiation. Using muscle tissue or SCs isolated from CBA or sucrose fed SIV‐infected rhesus macaques, we demonstrate that there is a significant increase in the expression of TGF‐β1 expression in the muscle and decreased myogenic differentiation of SCs of the CBA‐SIV animals. There was also increased expression of Smad2 in the CBA‐SIV SCs. The results provide evidence of dysregulation of the skeletal muscle milieu due to chronic alcohol in SIV infection that impairs SC myogenic differentiation.Grant Funding Source: NIH P60AA09803

Age-dependent expression of Pten and Smad4 genes in the urogenital system of Wistar rats.

To analyze Pten and Smad4 gene expression in the urogenital system of Wistar rats in differents ages. Pten and Smad4 mRNA expression was assessed in the bladder, ventral prostate, testis, ovaries, and uterus by real-time PCR. Statistical analysis using the ANOVA (p<0.05). Pten levels showed a progressive age-dependent increase in the bladder (male and female) and prostate and were elevated in the ovaries of the middle-aged. In the uterus, no statistically significant differences were observed; in the testis, increased and decreased levels were seen in young adult and middle-aged rats, respectively. Smad4 expression was downregulated in the ovaries of the pubertal group but increased in the middle age group. In the uterus, Smad4 expression in the oldest group was higher than the others groups. In the testis, Smad4 expression steadily declined with age; in the prostate, it was higher in middle-aged rats than in younger rats. A similar trend was observed in the bladder of male and female middle-aged rats, compared with the pubertal group. The changes in phosphatase tensin homologue and Smad4 mRNA expression in Wistar rats appear to be associated with hormonal modifications in puberty and may be related to early follicular and testicular development.

Extragastric modulation of host iron metabolism genes in a mouse model of H. Pylori infection

Aim There is increasing evidence from clinical and population studies for a role of Helicobacter pylori infection in the aetiology of iron supplementation refractory iron deficiency anaemia. This study examines the expression of genes involved in iron metabolism, in particular the regulatory hormone hepcidin, within a mouse model of H. pylori infection. Methods C57B16 female mice were infected at 5 weeks of age with the mouse-adapted H. pylori strain SS1 via oral gavage. Gastric and liver tissue was harvested 20 weeks post infection, total RNA was extracted and complementary DNA was produced from the mRNA by reverse transcription. Semi-quantitative Real Time PCR was used to examine the relative gene expression of Ferro-portin (Fpn1), total hepcidin (tHamp) and upstream regulators Smad7 and Bmp6 in infected (n = 10) and uninfected controls (n = 10). Results In gastric tissue from H. pylori infected mice there was reduced expression of tHamp (p = 0.03) and Smad7 (p Conclusions: Mice infected with H. pylori showed a significantly increased relative hepcidin gene expression in both gastric and hepatic tissue when compared with uninfected controls. This provides evidence for the presence of H. pylori, a known gastric pathogen, to cause changes in extragastric expression of iron metabolism genes.

Inhibition of corneal fibrosis by Smad7 in rats after photorefractive keratectomy

Background Haze or corneal subepithelial fibrosis is one of the common complications after refractive surgery procedures, such as photorefractive keratectomy (PRK), laser epithelial keratomileusis, and epipolis laser in situ keratomileusis, which would result in refractive regression, decreased visual quality, and corneal opacification. Haze directly resulted from corneal fibrosis mediated by transforming growth factor β (TGFβ). Smad7, an inhibitory Smad, can inhibit TGFβ signal transduction. Recently, the effects of Smad7 on the inhibition of fibrosis in several organs have been studied, while little is known about the effects on cornea after PRK. This study was aimed to determine the effects of lentiviral-mediated Smad7 gene expression on corneal fibrosis in rats after PRK. Methods Four different experimental groups were established using right eyes of Sprague-Dawley rats. Thirty-two eyes underwent de-epithelialization only and served as a sham operation group (group 1). Ninety-six eyes underwent PRK operation and were further divided into group 2 (the PRK group) without lentivector administration, group 3 (the Lv-blank group) with control lentiviral vector without Smad7 administration, and group 4 (the Lv-Smad7 group) with Smad7 expressing lentiviral vector Smad7 administration. At 1 day, 1 week, 1 month, and 3 months after PRK, the transfection efficiency was determined by measuring the fluorescence signal as well as Smad7 protein and mRNA levels. Corneas were further processed for immunoblotting to assess the phosphorylation of Smad2 as a downstream event of TGFβ/Smad signaling. The expression of fibrotic markers, such as α-smooth muscle actin (α-SMA), Type III collagen (collagen III), and cell cycle-related marker Ki67, was measured by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Results Lentivirus-mediated exogenous Smad7 gene expression in rat corneal tissue resulted in reduced activation of TGFβ/Smad signaling caused by downregulation of phosphorylation of Smad2. Smad7 also downregulated the expression of TGF 2. Markers of cell proliferation and fibrosis, including Ki67, α-SMA, and collagen III, were inhibited by Smad7 up to 3 months after PRK operation. Conclusion Smad7 gene transfer inhibits fibrogenic responses of cornea in rats after PRK.

815 MOLECULAR STUDY OF PTEN AND SMAD4 GENES ON UROGENITAL SYSTEM OF THE RATS IN DIFFERENT AGES

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research (I)1 Apr 2013815 MOLECULAR STUDY OF PTEN AND SMAD4 GENES ON UROGENITAL SYSTEM OF THE RATS IN DIFFERENT AGES Beatriz Rocha, Sicília Colli, Leilane Nepomuceno, Diogo Souza, Bianca Gregório, Carla Gallo, and Francisco Sampaio Beatriz RochaBeatriz Rocha Rio de Janeiro, Brazil More articles by this author , Sicília ColliSicília Colli Rio de Janeiro, Brazil More articles by this author , Leilane NepomucenoLeilane Nepomuceno Rio de Janeiro, Brazil More articles by this author , Diogo SouzaDiogo Souza Rio de Janeiro, Brazil More articles by this author , Bianca GregórioBianca Gregório Rio de Janeiro, Brazil More articles by this author , Carla GalloCarla Gallo Rio de Janeiro, Brazil More articles by this author , and Francisco SampaioFrancisco Sampaio Rio de Janeiro, Brazil More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.381AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Cancer is increasing in low- and middle-income countries and it is a major public health problem in many parts of the world. Genes PTEN and Smad4 are involved in many cellular processes such as cell proliferation, migration, cell survival and apoptosis. Levels of Smad4 are increased in Gleason score 3-4 and are reduced in proportion to the increase of Gleason score. Recent studies showed that the combination of PTEN and Smad4 genes resulted in a high efficiency as a predictive tool of patient survival. We aimed to identify the expression of PTEN and Smad4 upon urogenital system organs of Wistar rats at different ages. METHODS The study was approved by the animal care and use committee of the Local Biology Institute (021/2012). Males and females wistar rats were separated into 3 groups, according to the age group corresponding: 21days (males, n=10 and females, n=5), 50 days (males, n= 5 and females, n=5) and 90 days (males, n=10 and females, n=5). All animals were killed with a lethal dose of pentobarbital. Bladder (males and females), ventral prostate, testis, ovary and uterus were removed and stored at −80°C for RNA extraction. Total RNA from tissue was extracted using trizol reagent, then 1 ug RNA was reverse transcribed by Superscript III to obtain cDNA. cDNA was used to evaluate the gene expression of PTEN and Smad4 by real time PCR using SYBR GreenER. The reactions were normalized by β-actin gene expression. Data were analyzed by one-way ANOVA and Bonferroni post-test, considering p<0.05. RESULTS Concerning PTEN gene expression in different ages no difference was found on bladder and prostate in male rats. On the other hand, the group of 90 days showed an increase of 80% in its expression on testis when compared to the 21 days group (p = 0.02). In different ages of females, PTEN gene expression did not vary on bladder and ovaries. As for gene expression of Smad4 in males, there were no differences in prostate and bladder among the groups. However, its expression was reduced on testis at 50 and 90 days (93% and 83%, respectively) when compared to the 21 days (p = 0.0005). As for females, the gene expression of Smad4 in bladder did not differ among different ages. Concerning ovary, the authors observed a reduction of 73% and 59% in its expression at 50 and 90 days, respectively, compared to the 21 days (p = 0.0030). CONCLUSIONS Thus, the increase in the expression of PTEN to 90 days can indicate a connection between the gender and its entry into puberty. Similarly, a reduction in the expression of Smad4 to 50 and 90 days seems to show an important role in the early development of the folicular and testicular development. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e335 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Beatriz Rocha Rio de Janeiro, Brazil More articles by this author Sicília Colli Rio de Janeiro, Brazil More articles by this author Leilane Nepomuceno Rio de Janeiro, Brazil More articles by this author Diogo Souza Rio de Janeiro, Brazil More articles by this author Bianca Gregório Rio de Janeiro, Brazil More articles by this author Carla Gallo Rio de Janeiro, Brazil More articles by this author Francisco Sampaio Rio de Janeiro, Brazil More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract 19616: Down-Regulation of miR-23/27/24 Clusters by TGF-β1 Signalling Induces Phenotypic Switching of Vascular Smooth Muscle Cells in vitro and in Response to Vascular Injury in vivo

Background: The differentiated state of SMCs plays a key role in the pathogenesis of several major human diseases, including atherosclerosis and restenosis after vascular injury. Several miRNAs have recently been implicated in vascular smooth muscle cells differentiation and/or phenotypic switching. Members of the miR-23/27/24 clusters are highly expressed in smooth muscle cells and are involved in cell cycle control, proliferation and differentiation of various cell types. However, the role of miR-23/27/24 clusters in vascular disease is currently unknown. Thus, the aim of the present study was to evaluate the role of miR-23/27/24 cluster on SMC differentiation and proliferation in vitro and after vascular injury in vivo. Methods and Results: To determine the expression changes of miRNAs in vascular wall with neointimal formation in vivo , we used the standard rat carotid artery balloon injury model. Using real-time PCR, we demonstrated for the first time that expression of miR-24 and miRNAs that are clustered with it (members of the miR-23 and miR-27 families) are consistently downregulated 3 and 7 days after vascular injury. Furthermore, in vitro studies showed that TGF-β1 down-regulated miR-23/27/24 clusters expression in a dose- and time-dependent manner. By using both gain-of-function and loss-of-function approaches, we found that members of the miR-23/27/24 clusters promote multiple aspects of vascular smooth muscle cell contractile phenotype and alter TGF-β1 pathway signalling. Inhibition of miR-23a/b increased gene expression of SMAD-2 and SMAD-3, while ectopic expression of miR-24 reduces the mRNA and protein levels of different genes that regulate cell-cycle progression. Also, using BrdU incorporation and cell migration assay, we demonstrate that miR-24 inhibits vascular smooth muscle cell proliferation and migration. Furthermore, systemic delivery of miR-24 mimics markedly decreased neointimal hyperplasia after balloon injury. Conclusions: We demonstrated that miR-23/27/24 clusters control TGF-β1 pathway signalling in vascular smooth muscle cell in vitro and in vivo . These findings suggest that members of the miR-23/27/24 clusters may have the potential to suppress neointimal hyperplasia after arterial injury.

MiR-146a Expression Level As a Molecular Marker in Acute Promyleocytic Leukemia

AbstractAbstract 4821MiR-146a which is located on chromosomes 5 may be involved in normal hemtopoisis and the pathogenesis of several hematopoietic diseases through inhibiting the expression of its targets. Acute promyelocytic leukemia (APL) cell line NB4 was taken as research model to find out whether miR-146a expression was associated to cell differentiation and proliferation. The miR-146a expression level of NB4 cells decreased after ATRA treatment, meanwhile the expression level of its predicted target gene Smad4 increased simultaneously.MiR-146a expression plasmid, no related negative control sequence plasmid and recombined luciferase reporter gene vector containing the miR-146a complementary combined site on Smad4 plasmid were constructed by DNA recombination technical. Both recombined plasmids were transiently co-transfected into 293T cells with control plasmid pRL-TK. The interaction between miR-146a and its predicted target gene Smad4 was confirmed by the relative activities of dual luciferase detection.This interaction relationship was further confirmed by liposome entrapment transfection of miR-146a and its antisense molecular in NB4 cells. The expression level of miR-146a in NB4 cells did no influence on Smad4 gene expression but Smad4 protein expression levels (fig 1).The changes of NB4 cells on biological behaviors were observed by cell morphology, proliferation curve and flow cytometry methods under different miR-146a expression status. The results showed that cell proliferation ratio decreased and cell apoptosis ratio increased after miR-146a expression suppression in NB4 cells.The investigation of miR-146a expression levels in mononuclear cells of APL patients' bone marrow and clinical data were carried out. It was showed that the expression levels of miR-146a were higher in APL patients than normal controls. The expression level of miR-146a was positive correlated with peripheral blood white cell count (fig 2). MiR-146a expression level was negative correlated with Th/Ts ratio among these patients (fig 3).MiR-146a/Smad4 signal pathway is expressed in acute promyelocytic leukemia. The proliferation and apoptosis of the cells are modified by the Smad4 protein expression regulated by miR-146a expression. MiR-146a expression is somewhat associated with acknowledged clinical prognosis factors. It has potential possibility to take miR-146a as prognosis factor. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Investigation of the skin repair and healing mechanism of N‐carboxymethyl chitosan in second‐degree burn wounds

N-carboxymethyl chitosan (NCMC) was synthesized with the modification of chitosan; the substitution degree was measured by titration. The biocompatibility and degradability of the NCMC were studied in vivo and the results showed that the NCMC was nontoxic and biocompatible. The in vivo degradation rate of NCMC in musculature was faster than that in subcutaneous tissue due to the relatively high lysozyme concentration. The NCMC was used as biomaterial to heal deep second-degree burn wounds. The wound size reduction, histological examination, and the quantification of transforming growth factor-β(1) , tumor necrosis factor-α and interleukin-8 protein levels, and Smad3 gene expression were measured to evaluate the healing effects. The results demonstrated that the NCMC was efficient in accelerating wound healing via activating transforming growth factor-β(1) /Smad3 signaling pathway.

Effects of RNA interference-induced Smad3 gene silencing on pulmonary fibrosis caused by paraquat in mice

Paraquat (PQ) poisoning induces many physiological and histological changes in the human body, but PQ-induced pulmonary fibrosis is most often associated with death. The signaling pathway associated with pulmonary fibrosis is reliant on transforming growth factor-beta 1 (tgf-β(1)) activation of Smad3, as evidenced by Smad3-deficient mice being resistant to tgf-β(1)-induced pulmonary fibrosis. Thus, we sought to determine whether targeted silencing of Smad3 gene expression could inhibit PQ-induced pulmonary fibrosis in mice. We developed an RNA interference (RNAi) method using short hairpin RNAs (shRNAs) targeting Smad3. The shRNA expression cassettes capable of effectively silencing Smad3 in L929 mouse fibroblasts were transferred to an adenovirus vector and intratracheally administered into mouse lung. Treated mice presented with inhibited Smad3 mRNA and protein and were resistant to PQ-induced pulmonary fibrosis, as evidenced by suppressed expressions of procollagen type I mRNA and hydroxyproline amino acid. Thus, silencing of Smad3 appears to be a promising alternative strategy for the treatment of PQ-induced pulmonary fibrosis.

Strength Training with Blood Flow Restriction Diminishes Myostatin Gene Expression

The aim of the study was to determine whether the similar muscle strength and hypertrophy responses observed after either low-intensity resistance exercise associated with moderate blood flow restriction or high-intensity resistance exercise are associated with similar changes in messenger RNA (mRNA) expression of selected genes involved in myostatin (MSTN) signaling. Twenty-nine physically active male subjects were divided into three groups: low-intensity (20% one-repetition maximum (1RM)) resistance training (LI) (n = 10), low-intensity resistance exercise associated with moderate blood flow restriction (LIR) (n = 10), and high-intensity (80% 1RM) resistance exercise (HI) (n = 9). All of the groups underwent an 8-wk training program. Maximal dynamic knee extension strength (1RM), quadriceps cross-sectional area (CSA), MSTN, follistatin-like related genes (follistatin (FLST), follistatin-like 3 (FLST-3)), activin IIb, growth and differentiation factor-associated serum protein 1 (GASP-1), and MAD-related protein (SMAD-7) mRNA gene expression were assessed before and after training. Knee extension 1RM significantly increased in all groups (LI = 20.7%, LIR = 40.1%, and HI = 36.2%). CSA increased in both the LIR and HI groups (6.3% and 6.1%, respectively). MSTN mRNA expression decreased in the LIR and HI groups (45% and 41%, respectively). There were no significant changes in activin IIb (P > 0.05). FLST and FLST-3 mRNA expression increased in all groups from pre- to posttest (P < 0.001). FLST-3 expression was significantly greater in the HI when compared with the LIR and LI groups at posttest (P = 0.024 and P = 0.018, respectively). GASP-1 and SMAD-7 gene expression significantly increased in both the LIR and HI groups. We concluded that LIR was able to induce gains in 1RM and quadriceps CSA similar to those observed after traditional HI. These responses may be related to the concomitant decrease in MSTN and increase in FLST isoforms, GASP-1, and SMAD-7 mRNA gene expression.

MiR-30 Family Members Negatively Regulate Osteoblast Differentiation

miRNAs are endogenously expressed 18- to 25-nucleotide RNAs that regulate gene expression through translational repression by binding to a target mRNA. Recently, it has been indicated that miRNAs are closely related to osteogenesis. Our previous data suggested that miR-30 family members might be important regulators during the biomineralization process. However, whether and how they modulate osteogenic differentiation have not been explored. In this study, we demonstrated that miR-30 family members negatively regulate BMP-2-induced osteoblast differentiation by targeting Smad1 and Runx2. Evidentially, overexpression of miR-30 family members led to a decrease of alkaline phosphatase activity, whereas knockdown of them increased the activity. Then bioinformatic analysis identified potential target sites of the miR-30 family located in the 3' untranslated regions of Smad1 and Runx2. Western blot analysis and quantitative RT-PCR assays demonstrated that miR-30 family members inhibit Smad1 gene expression on the basis of repressing its translation. Furthermore, dual-luciferase reporter assays confirmed that Smad1 is a direct target of miR-30 family members. Rescue experiments that overexpress Smad1 and Runx2 significantly eliminated the inhibitory effect of miR-30 on osteogenic differentiation and provided strong evidence that miR-30 mediates the inhibition of osteogenesis by targeting Smad1 and Runx2. Also, the inhibitory effects of the miR-30 family were validated in mouse bone marrow mesenchymal stem cells. Therefore, our study uncovered that miR-30 family members are key negative regulators of BMP-2-mediated osteogenic differentiation.

Effects of soybean peptide and collagen peptide on collagen synthesis in normal human dermal fibroblasts

The collagen present in the dermis of the skin is a fibrous protein that fills the gaps between cells and helps maintain tissue flexibility. Effectively increasing the collagen present in the skin is an important goal for cosmetic research. Recent research has shown that soybean peptide (SP) has anti-fatigue activity, antioxidant activity, and the ability to increase type I collagen, while collagen peptide (CP) has the ability to enhance corneal moisture content and viscoelasticity, as well as to increase levels of hyaluronic acid synthesizing enzymes in human skin. Little documented research, however, has been conducted on collagen formation in relation to these peptides. Therefore, this research applied SP and CP with molecular weights primarily around 500 and preparations containing both SP and CP to normal human dermal fibroblasts together with magnesium ascorbyl phosphate (VC-PMg), and used real-time PCR to determine the gene expression of type I collagen (COL1A1), which contributes to collagen synthesis, and Smad7, which contribute to collagen breakdown. In addition, enzyme linked immuno sorbent assay (ELISA) was used to measure collagen content in the media. COL1A1 gene expression at 24 h after sample addition showed higher tendency in all samples and increased with time at 4, 8 and 24 h after addition. Smad7 gene expression was not substantially different at 4 h after addition. matrix metalloproteinase-1 gene expression was higher following SP addition, but was lower after the addition of CP and SP+CP. Medium collagen content was higher in all samples and increased with time at 8 h after addition. Collagen levels were higher when SP and CP were added together.

Smad1 and WIF1 genes are downregulated during saccular stage of lung development in the nitrofen rat model

The exact pathogenesis of pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia (CDH) still remains unclear. Smad1, one of the bone morphogenesis protein (BMP) receptor downstream signaling proteins, plays a key role in organogenesis including lung development and maturation. Smad1 knockout mice display reduced sacculation, an important feature of pulmonary hypoplasia. Wnt inhibitor factor 1 (Wif1) is a target gene of Smad1 in the developing lung epithelial cells (LECs). Smad1 directly regulates Wif1 gene expression and blockade of Smad1 function in fetal LECs is reported to downregulate Wif1 gene expression. We designed this study to test the hypothesis that pulmonary Smad1 and Wif1 gene expression is downregulated during saccular stage of lung development in the nitrofen CDH model. Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetuses were harvested on D18, and D21. Fetal lungs were dissected and divided into 2 groups: control and nitrofen (n = 9 at each time point, respectively). Pulmonary gene expression of Smad1 and Wif1 were analyzed by real-time RT-PCR. Immunohistochemistry was performed to evaluate protein expression/distribution of Smad1 and Wif1. The relative mRNA expression levels of Smad1 and Wif1 were significantly downregulated in the nitrofen group compared to controls on D18 and D21 (*p < 0.01, **p < 0.05). Immunoreactivity of Smad1 and Wif1 was also markedly decreased in nitrofen lungs compared to controls on D18 and D21. We provide evidence, for the first time, that the pulmonary gene expression of Smad1 and Wif1 is downregulated on D18 and D21 (saccular stage of lung development) in the nitrofen-induced hypoplastic lung. These findings suggest that the downregulation of Smad1/Wif1 gene expression may contribute to pulmonary hypoplasia in the nitrofen CDH model by retardation of lung development during saccular stage.

The effect of Smad3 knockdown on the secretion of extracellular matrix induced by IGFBPrP1 in activated hepatic stellate cells

Objective To identify the effect of IGFBPrP1 on the secretion of extracellular matrix in hepatic stellate cells through the Smad3 signaling pathway. Methods (1)Two pairs of chemically synthesized siRNAs (siRNA1, siRNA2) targeting Smad3 were transfected into HSC-T6 cells,real-time PCR and Western blot were used to evaluate the silence efficiency, and the better siRNA was used. (2)HSC-T6 cells were divided into four groups: Negative control group, siRNA-Smad3 transfection group, siRNA-Smad3+IGFBPrP1 group and IGFBPrP1 group. The better siRNA was chosen to transfect into HSC-T6 cells. The protein expressions of Smad3, fibronectin and Collagen Ⅰ were evaluated by Western blot. Results (1)siRNA2-Smad3 inhibited Smad3 gene expression stronger than another siRNA. (2)After transfection of siRNA2-Smad3, the protein expression of Smad3 was significantly decreased compared to the negative control group(P＜0.01). The protein expression of fibronectin and Collagen Ⅰ in IGFBPrP1 stimulating HSCs treated with siRNA2-Smad3 were significantly decreased compared to that in IGFBPrP1 stimulating HSC without siRNA2-Smad3 (P ＜0. 01 ).Conclusion IGFBPrP1 induces the secretion of extracellular matrix in hepatic stellate cells through the Smad3 signaling pathway. Key words: Insulin-like growth factor binding protein related protein1; RNA interference; Smad3; Hepatic stellate cells; Hepatic fibrosis

MicroRNA-26a is a novel regulator of vascular smooth muscle cell function.

Aberrant smooth muscle cell (SMC) plasticity has been implicated in a variety of vascular disorders including atherosclerosis, restenosis, and abdominal aortic aneurysm (AAA) formation. While the pathways governing this process remain unclear, epigenetic regulation by specific microRNAs (miRNAs) has been demonstrated in SMCs. We hypothesized that additional miRNAs might play an important role in determining vascular SMC phenotype. Microarray analysis of miRNAs was performed on human aortic SMCs undergoing phenotypic switching in response to serum withdrawal, and identified 31 significantly regulated entities. We chose the highly conserved candidate miRNA-26a for additional studies. Inhibition of miRNA-26a accelerated SMC differentiation, and also promoted apoptosis, while inhibiting proliferation and migration. Overexpression of miRNA-26a blunted differentiation. As a potential mechanism, we investigated whether miRNA-26a influences TGF-β-pathway signaling. Dual-luciferase reporter assays demonstrated enhanced SMAD signaling with miRNA-26a inhibition, and the opposite effect with miRNA-26a overexpression in transfected human cells. Furthermore, inhibition of miRNA-26a increased gene expression of SMAD-1 and SMAD-4, while overexpression inhibited SMAD-1. MicroRNA-26a was also found to be downregulated in two mouse models of AAA formation (2.5- to 3.8-fold decrease, P < 0.02) in which enhanced switching from contractile to synthetic phenotype occurs. In summary, miRNA-26a promotes vascular SMC proliferation while inhibiting cellular differentiation and apoptosis, and alters TGF-β pathway signaling. MicroRNA-26a represents an important new regulator of SMC biology and a potential therapeutic target in AAA disease.

Quantitative analysis of transient and sustained transforming growth factor‐β signaling dynamics

Mammalian cells can decode the concentration of extracellular transforming growth factor-β (TGF-β) and transduce this cue into appropriate cell fate decisions. How variable TGF-β ligand doses quantitatively control intracellular signaling dynamics and how continuous ligand doses are translated into discontinuous cellular fate decisions remain poorly understood. Using a combined experimental and mathematical modeling approach, we discovered that cells respond differently to continuous and pulsating TGF-β stimulation. The TGF-β pathway elicits a transient signaling response to a single pulse of TGF-β stimulation, whereas it is capable of integrating repeated pulses of ligand stimulation at short time interval, resulting in sustained phospho-Smad2 and transcriptional responses. Additionally, the TGF-β pathway displays different sensitivities to ligand doses at different time scales. While ligand-induced short-term Smad2 phosphorylation is graded, long-term Smad2 phosphorylation is switch-like to a small change in TGF-β levels. Correspondingly, the short-term Smad7 gene expression is graded, while long-term PAI-1 gene expression is switch-like, as is the long-term growth inhibitory response. Our results suggest that long-term switch-like signaling responses in the TGF-β pathway might be critical for cell fate determination.

Structural and functional analysis of SMAD4 gene promoter in malignant pancreatic and colorectal tissues: Detection of two novel polymorphic nucleotide repeats

Background: The tumor suppressor gene SMAD4 (DPC4) encodes for the common intracellular mediator of the TGF-β superfamily pathway, which regulates numerous cellular processes, such as cell proliferation, cell differentiation, apoptosis, cell fate and migration. This study was aimed to investigate the presence of genetic variants in SMAD4 gene promoter in malignant pancreatic and colorectal tissue and to analyze their functional consequences. Methods: The study was performed on genomic DNA isolated from malignant tissue samples obtained on surgery from 50 patients with pancreatic carcinoma and 50 patients with colorectal cancer. Screening for mutations within an 800 bp-long fragment of the SMAD4 gene promoter was performed by DNA sequencing and two mononucleotide repeats, at positions −462 and −4, were found to be polymorphic in malignant tissue. The exact number of thymidines in the tracts −462T(15) and −4T(12) was determined by PCR with fluorescently labeled primers followed by capillary electrophoresis. Functional analysis of −462T(15)/−4T(12) haplotypes was performed by luciferase reporter assays. Results: Haplotype −462T(14)/−4T(10) was found in 85% of pancreatic cancer tissues, but it was not present in any of colorectal cancer tissues. Statistically significant reduction ( p < 0.001) in activity was observed in the haplotype −462T(14)/−4T(10) in comparison with the haplotypes −462T(15)/−4T(12) and −462T(14)/−4T(11). Conclusion: Results of this study indicate that novel genetic variant −4T(10) in the SMAD4 gene promoter affects its activity and that element −4T(12) may play a role in transcriptional regulation of SMAD4 gene expression. Obtained results, though preliminary, also indicate that SMAD4 gene promoter haplotype −462T(14)/−4T(10) may represent a genetic marker of potential relevance for pancreatic and colorectal cancer. The findings of this study should be confirmed by further investigation in these two and other tumors, on larger number of patients and with different tumor stages. Translational research aimed at investigating potential application of mononucleotide repeats −462T(15) and −4T(12) in SMAD4 gene promoter as molecular markers in cancer may also prove useful.