BACKGROUND AND OBJECTIVES: There have been contradictory reports on the pro-cancer or anti-cancer effects of mesenchymal stem cells. In this study, we investigated whether conditioned medium (CM) from hypoxic human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) (H-CM) showed enhanced anti-cancer effects compared with CM from normoxic hUC-MSCs (N-CM). METHODS AND RESULTS: Compared with N-CM, H-CM not only strongly reduced cell viability and increased apoptosis of human cervical cancer cells (HeLa cells), but also increased caspase-3/7 activity, decreased mitochondrial membrane potential (MMP), and induced cell cycle arrest. In contrast, cell viability, apoptosis, MMP, and cell cycle of human dermal fibroblast (hDFs) were not significantly changed by either CM whereas caspase-3/7 activity was decreased by H-CM. Protein antibody array showed that activin A, Beta IG-H3, TIMP-2, RET, and IGFBP-3 were upregulated in H-CM compared with N-CM. Intracellular proteins that were upregulated by H-CM in HeLa cells were represented by apoptosis and cell cycle arrest terms of biological processes of Gene Ontology (GO), and by cell cycle of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In hDFs, negative regulation of apoptosis in biological process of GO and PI3K-Akt signaling pathway of KEGG pathways were represented. CONCLUSIONS: H-CM showed enhanced anti-cancer effects on HeLa cells but did not influence cell viability or apoptosis of hDFs and these different effects were supported by profiling of secretory proteins in both kinds of CM and intracellular signaling of HeLa cells and hDFs.
Activins ; Anoxia ; Apoptosis ; Biological Processes ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Survival ; Culture Media, Conditioned ; Fibroblasts ; Gene Ontology ; Genome ; HeLa Cells ; Humans ; Insulin-Like Growth Factor Binding Protein 3 ; Membrane Potential, Mitochondrial ; Mesenchymal Stromal Cells ; Tissue Inhibitor of Metalloproteinase-2 ; Uterine Cervical Neoplasms

Insulin-like growth factor binding proteins (IGFBPs) are major regulators of insulin-like growth factor bioavailability and activity in metabolic signaling. Seven IGFBP family isoforms have been identified. Recent studies have shown that IGFBPs play a pivotal role in metabolic signaling and disease, including the pathogenesis of obesity, diabetes, and cancer. Although many studies have documented the various roles played by IGFBPs, transcriptional regulation of IGFBPs is not well understood. In this review, we focus on the regulatory mechanisms of IGFBP gene expression, and we summarize the findings of transcription factor activity in the IGFBP promoter region.
Biological Availability ; Gene Expression ; Humans ; Insulin-Like Growth Factor Binding Protein 2* ; Insulin-Like Growth Factor Binding Proteins ; Liver ; Metabolic Diseases ; Obesity ; Promoter Regions, Genetic ; Protein Isoforms ; Transcription Factors

Angiotensin I ; Hepatic Stellate Cells ; Insulin-Like Growth Factor Binding Protein 2 ; Peptide Fragments ; Peptidyl-Dipeptidase A

OBJECTIVETo investigate the effect of inositol hexaphosphate (IP6) on proliferation of human prostate carcinoma LNCaP cells and its relation to insulin-like growth factors binding protein-3 (IGFBP-3) expression.

METHODSThe siRNA technology was used to silence the IGFBP-3 gene in LNCaP cells. LNCaP cells and IGFBP-3 gene silenced LNCaP cells were exposed to IP6 for 24 h. Cell viability was measured by MTT assay; cell cycle arrest and cell apoptosis were detected by flow cytometry. The expression levels of IGFBP-3 and Bcl-2 mRNA and protein were analyzed by real-time quantitative RT-PCR and Western blotting, respectively.

RESULTSThe proliferation of LNCaP cells was be inhibited by IP6 in a dose dependent manner. After exposure to IP6 for 24 h, the cell viability in LNCaP cells and siRNA-treated LNCaP cells was 53.2%±11.6% and 82.3%±10.9%, respectively (P<0.05). After treatment of 1.5 mmol IP6，the apoptosis rate of LNCAP cells and siRNA-treated LNCAP cells was 40.48%±13.21% and 30.43%±10.65%, respectively (P<0.05). The proportion of G1 and G2 phase in LNCAP cells was 70.58%±8.25% and 5.64%±1.23%，after IP6 treatment the percentage of G1 phase cells decreased to 48.66%±11.23% and G2 phase cells increased to 31.11%±9.68%. However, for siRNA treated LNCAP cells, the proportion of G1 phase cells was 58.25%±12.36% and G2 phase cells was 23.85%±12.45%. Higher expression of IGFBP-3 and lower expression of Bcl-2 in LNCaP cells treated with IP6 were found at both mRNA and protein levels. IP6 treatment enhanced IGFBP-3 mRNA expression by 2.21±0.15 folds. In the contrast, expression of Bcl-2 mRNA decreased by 0.69±0.03 folds. Meanwhile, after IGFBP- gene silence Bcl-2 expression was not decreased.

CONCLUSIONIP6 can inhibit the proliferation of LNCaP cells, which may be associated with the changes of IGFBP-3 level through Bcl-2 expression.

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Gene Silencing ; Humans ; Insulin-Like Growth Factor Binding Protein 3 ; metabolism ; Male ; Phytic Acid ; pharmacology ; Prostatic Neoplasms ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Small Interfering

Cows with different Insulin-like Growth Factor-I (IGF-I) concentrations showed comparable expression levels of hepatic growth hormone receptor (GHR). Suppressor of cytokine signaling 2 (SOCS2), could be responsible for additional inhibition of the GHR signal cascade. The aims were to monitor cows with high or low antepartal IGF-I concentrations (IGF-I(high) or IGF-I(low)), evaluate the interrelationships of endocrine endpoints, and measure hepatic SOCS2 expression. Dairy cows (n = 20) were selected (240 to 254 days after artificial insemination (AI)). Blood samples were drawn daily (day -17 until calving) and IGF-I, GH, insulin, thyroid hormones, estradiol, and progesterone concentrations were measured. Liver biopsies were taken (day 264 +/- 1 after AI and postpartum) to measure mRNA expression (IGF-I, IGFBP-2, IGFBP-3, IGFBP-4, acid labile subunit (ALS), SOCS2, deiodinase1, GHR1A). IGF-I concentrations in the two groups were different (p < 0.0001). However, GH concentrations and GHR1A mRNA expression were comparable (p > 0.05). Thyroxine levels and ALS expression were higher in the IGF-I(high) cows compared to IGF-I(low) cows. Estradiol concentration tended to be greater in the IGF-I(low) group (p = 0.06). It was hypothesized that low IGF-I levels are associated with enhanced SOCS2 expression although this could not be decisively confirmed by the present study.
Animals ; Cattle ; Estradiol/blood ; Female ; Growth Hormone/blood ; Insulin/blood ; Insulin-Like Growth Factor Binding Protein 2/analysis ; Insulin-Like Growth Factor Binding Protein 3/analysis ; Insulin-Like Growth Factor Binding Protein 4/analysis ; Insulin-Like Growth Factor I/*analysis/physiology ; Liver/chemistry ; Pregnancy/metabolism/physiology ; Pregnancy, Animal/*metabolism/physiology ; Progesterone/blood ; Suppressor of Cytokine Signaling Proteins/analysis ; Thyroid Hormones/blood

Insulin-like growth factor binding protein (IGFBP)-3 has roles in modulating the effect of IGFs by binding to IGFs and inhibiting cell proliferation in an IGF-independent manner. Although recent studies have been reported that IGFBP-3 has also roles in metabolic regulation, their exact roles in adipose tissue are poorly understood. In this review, we summarized the studies about the biological roles in glucose and lipid metabolism. IGFBP-3 overexpression in transgenic mice suggested that IGFBP-3 results in glucose intolerance, and insulin resistance. IGFBP-3 knockout (KO) mice exhibited normal insulin level and glucose response after glucose challenge. More recent study in IGFBP-3 KO mice with a high-fat diet demonstrated that IGFBP-3 KO mice exhibited elevated fasting glucose and insulin, but normal response to glucose challenge, suggesting that IGFBP-3 KO mice may induce insulin resistance even though preserved insulin sensitivity. In vitro and in vivo studies using 3T3-L1 adipocytes and rat, IGFBP-3 induced insulin resistance by inhibiting glucose uptake. In contrast, the reduced levels of IGFBP-3 in obesity might induce insulin resistance by suppression of IGFBP-3's anti-inflammatory function, suggesting IGFBP-3 has a protective effect on insulin resistance. Also, proteolysis of IGFBP-3 might contribute to the insulin resistance in obesity and type 2 diabetes mellitus. In addition, IGFBP-3 inhibited adipocyte differentiation, suggesting IGFBP-3 may contribute to the insulin insensitivity. Taken together, it is not yet certain that IGFBP-3 has a protective effect or enhancing effect on insulin resistance, and more studies will be needed to clarify the roles of IGFBP-3 in metabolic regulation.
Adipocytes ; Adipose Tissue ; Animals ; Carrier Proteins ; Cell Proliferation ; Diabetes Mellitus, Type 2 ; Diet, High-Fat ; Fasting ; Glucose ; Glucose Intolerance ; Insulin ; Insulin Resistance ; Insulin-Like Growth Factor Binding Protein 3 ; Lipid Metabolism ; Mice ; Mice, Transgenic ; Obesity ; Proteolysis ; Rats

BACKGROUNDSterol regulatory element binding protein (SREBP)-2 plays a key role in lipid homeostasis by stimulating gene expression of cholesterol biosynthetic pathways. The insulin-like growth factor binding protein (IGFBP) family regulates growth and metabolism, especially bone cell metabolism, and correlates with osteonecrosis. However, association of their gene polymorphisms with risk of avascular necrosis of the femoral head (ANFH) has rarely been reported. We determined whether SREBP-2 and IGFBP-3 gene polymorphisms were associated with increased ANFH risk in the Chinese population.

METHODSTwo single nucleotide polymorphisms of SREBP2 gene, rs2267439 and rs2267443, and one of IGFBP-3 gene, rs2453839, were selected and genotyped in 49 ANFH patients and 42 control individuals by direct sequencing assay.

RESULTSThe frequencies of rs2267439 TT and rs2267443 GA of SREBP2 and rs2453839 TT and CT of IGFBP-3 in the ANFH group showed increased and decreased tendencies (against normal control group), respectively. Interaction analysis of genes revealed that the frequency of carrying rs2267439 TT and rs2267443 GA genotypes of SREBF-2 in ANFH patients was significantly higher than in the control group (P < 0.05). Association analysis between polymorphisms and clinical phenotype demonstrated that the disease course in ANFH patients with the rs2453839 TT genotype of IGFBP-3 was significantly shorter than that of CT + CC carriers (P < 0.01). CT + CC genotype frequency in patients with stage III/IV bilateral hip lesions was significantly higher than in those with stage III/IV unilateral lesions and stage II/III bilateral lesions (P < 0.05 - 0.02).

CONCLUSIONSOur results suggested that interaction of SREBP-2 gene polymorphisms and the relationship between the polymorphisms and clinical phenotype of IGFBP-3 were closely related to increased ANFH risk in the Chinese population. The most significant finding was that the CT + CC genotype carriers of IGFBP-3 rs2453839 were highly associated with the development of ANFH.

Adult ; Asian Continental Ancestry Group ; genetics ; Female ; Femur Head Necrosis ; genetics ; Genetic Predisposition to Disease ; Humans ; Insulin-Like Growth Factor Binding Protein 3 ; genetics ; Male ; Middle Aged ; Polymerase Chain Reaction ; Polymorphism, Genetic ; genetics ; Sterol Regulatory Element Binding Protein 2 ; genetics

Angiotensin I ; metabolism ; Animals ; Insulin-Like Growth Factor Binding Protein 2 ; metabolism ; Liver ; metabolism ; pathology ; Liver Cirrhosis, Experimental ; metabolism ; pathology ; Peptide Fragments ; metabolism ; Peptidyl-Dipeptidase A ; metabolism ; Rats ; Rats, Wistar

PURPOSE: IGFBP-3 leads to the induction of insulin resistance in 3T3-L1 adipocytes. We carried out a series of experiments to elucidate the effects of IGFBP-3 on adipokines and gene expressions. METHODS: We treated fully-differentiated 3T3-L1 adipocytes with IGFBP-3 (0.5, 1, and 2 microg/mL) for one day and measured the mRNA levels of adiponectin, leptin, resistin, and TNF-alpha by RT-PCR, and adiponectin, leptin, resistin, and IL-6 protein levels in the culture supernatant were measured using multiplex adipokine assay ELISA Kits (Linco Research, St. Charles, Missouri). Gene expression in 3T3-L1 adipocyte cells using a microarray method was performed. RESULTS: IGFBP-3 inhibited the expression of adiponectin, leptin, resistin, and TNF-alpha mRNA. IGFBP-3 at 0.5 and 1 micro/mL decreased adiponectin release, but IL-6 release was increased at 2 micro/mL IGFBP-3. A dose-dependent inhibition of leptin was released by IGFBP-3 at 50%. Resistin release was decreased by 40%. The effect of IGFBP-3 on the gene expression in 3T3-L1 adipocyte cells using a microarray assay related to an increase of agouti-realted proteins (Agrp) and Janus kinase 2 (JAK2), and a decrease of the ras homolog gene family (Rhoq), acyl-CoA synthetase long-chain family member 6 (Acsl6), and the interleukin-1 receptor-associated kinase 1 (Irak1). CONCLUSION: IGFBP-3 regulates several adipokines gene expressions that are known to modulate insulin sensitivity, and this regulation may be attributable to the insulin resistance effect of IGFBP-3 on adipocytes.
Adipocytes ; Adipokines ; Adiponectin ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Insulin Resistance ; Insulin-Like Growth Factor Binding Protein 3 ; Interleukin-1 Receptor-Associated Kinases ; Interleukin-6 ; Janus Kinase 2 ; Leptin ; Ligases ; Proteins ; Resistin ; RNA, Messenger ; Tumor Necrosis Factor-alpha

AIMUnderstanding the response of mesenchymal stem cells (MSCs) to mechanical strain and their consequent gene expression patterns will broaden our knowledge of the mechanobiology of distraction osteogenesis.

METHODOLOGYIn this study, a single period of cyclic mechanical stretch (0.5 Hz, 2,000 microepsilon) was performed on rat bone marrow MSCs. Cellular proliferation and alkaline phosphatase (ALP) activity was examined. The mRNA expression of six bone-related genes (Ets-1, bFGF, IGF-II, TGF-beta, Cbfa1 and ALP) was detected using real-time quantitative RT-PCR.

RESULTSThe results showed that mechanical strain can promote MSCs proliferation, increase ALP activity, and up-regulate the expression of these genes. A significant increase in Ets-1 expression was detected immediately after mechanical stimulation, but Cbfa1 expression became elevated later. The temporal expression pattern of ALP coincided perfectly with Cbfa1.

CONCLUSIONThe results of this study suggest that mechanical strain may act as a stimulator to induce differentiation of MSCs into osteoblasts, and that these bone-related genes may play different roles in the response of MSCs to mechanical stimulation.

Alkaline Phosphatase ; analysis ; Animals ; Antigens, Surface ; analysis ; Biomechanical Phenomena ; Bone Marrow Cells ; physiology ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cell Proliferation ; Core Binding Factor Alpha 1 Subunit ; analysis ; Fibroblast Growth Factor 2 ; analysis ; Insulin-Like Growth Factor II ; analysis ; Mesenchymal Stromal Cells ; physiology ; Osteoblasts ; physiology ; Osteogenesis, Distraction ; Pluripotent Stem Cells ; physiology ; Proto-Oncogene Protein c-ets-1 ; analysis ; Rats ; Stress, Mechanical ; Transforming Growth Factor beta ; analysis ; Up-Regulation ; physiology