Characterization of increased mucus production of HT29-MTX-E12 cells grown under Semi-Wet interface with Mechanical Stimulation.

The intestinal mucus layer plays a crucial role in human health. To study intestinal mucus function and structure in vitro, the mucus-producing intestinal cell line HT29-MTX-E12 has been commonly used. However, this cell line produces only low amounts of the intestine-specific MUC2. It has been shown previously that HT29-MTX-E12 cells cultured under Semi-Wet interface with Mechanical Stimulation (SWMS) produced higher amounts of MUC2, concomitant with a thicker mucus layer, compared to cells cultured conventionally. However, it remains unknown which underlying pathways are involved. Therefore, we aimed to further explore the cellular processes underlying the increased MUC2 production by HT29-MTX-E12 cells grown under SWMS conditions. Cells grown on Transwell membranes for 14 days under static and SWMS conditions (after cell seeding and attachment) were subjected to transcriptome analysis to investigate underlying molecular pathways at gene expression level. Caco-2 and LS174T cell lines were included as references. We characterized how SWMS conditions affected HT29-MTX-E12 cells in terms of epithelial barrier integrity, by measuring transepithelial electrical resistance, and cell metabolism, by monitoring pH and lactate production per molecule glucose of the conditioned medium. We confirmed higher MUC2 production under SWMS conditions at gene and protein level and demonstrated that this culturing method primarily stimulated cell growth. In addition, we also found evidence for a more aerobic cell metabolism under SWMS, as shown previously for similar models. In summary, we suggest different mechanisms by which MUC2 production is enhanced under SWMS and propose potential applications of this model in future studies.

Bacterial folate biosynthesis and colorectal cancer risk: more than just a gut feeling.

Folate is a B-vitamin with an important role in health and disease. The optimal folate status with regard to human health remains controversial. A low intake of natural folate as well as excessive intake of synthetic folic acid, were previously linked to an increased risk of colorectal cancer or with aberrant molecular pathways related to carcinogenesis in some studies. Importantly, most studies conducted so far, solely focused on dietary intake or circulating levels of folate in relation to cancer risk. Notably, diet or dietary supplements are not the only sources of folate. Several bacteria in the gastrointestinal tract can synthesize B-vitamins, including folate, in quantities that resemble dietary intake. The impact of bacterial folate biosynthesis concerning human health and disease remains unexplored. This review highlights current insights into folate biosynthesis by intestinal bacteria and its implications for processes relevant to cancer development, such as epigenetic DNA modifications and DNA synthesis. Moreover, we will reflect on the emerging question whether food-grade or intestinal bacteria can be considered a potential target to ensure sufficient levels of folate in the gastrointestinal tract and, hence the relevance of bacterial folate biosynthesis for disease prevention or treatment.

Lifelong calorie restriction affects indicators of colonic health in aging C57Bl/6J mice.

Diminished colonic health is associated with various age-related pathologies. Calorie restriction (CR) is an effective strategy to increase healthy lifespan, although underlying mechanisms are not fully elucidated. Here, we report the effects of lifelong CR on indicators of colonic health in aging C57Bl/6J mice. Compared to an ad libitum control and moderate-fat diet, 30% energy reduction was associated with attenuated immune- and inflammation-related gene expression in the colon. Furthermore, expression of genes involved in lipid metabolism was higher upon CR, which may point towards efficient regulation of energy metabolism. The relative abundance of bacteria considered beneficial to colonic health, such as Bifidobacterium and Lactobacillus, increased in the mice exposed to CR for 28 months as compared to the other diet groups. We found lower plasma levels of interleukin-6 and lower levels of various metabolites, among which are bile acids, in the colonic luminal content of CR-exposed mice as compared to the other diet groups. Switching from CR to an ad libitum moderate-fat diet at old age (24 months) revealed remarkable phenotypic plasticity in terms of gene expression, microbiota composition and metabolite levels, although expression of a subset of genes remained CR-associated. This study demonstrated in a comprehensive way that CR affects indicators of colonic health in aging mice. Our findings provide unique leads for further studies that need to address optimal and feasible strategies for prolonged energy deprivation, which may contribute to healthy aging.

Association between DNA methylation profiles in leukocytes and serum levels of persistent organic pollutants in Dutch men.

Consumption of polluted fish may lead to high levels of persistent organic pollutants (POPs) in humans, potentially causing adverse health effects. Altered DNA methylation has been suggested as a possible contributor to a variety of adverse health effects. The aim of this study was to evaluate the relationship between serum POP levels (dioxins, polychlorobiphenyls, and perfluoroctane sulphonate) and DNA methylation. We recruited a total of 80 Dutch men who regularly consumed eel from either low- or high-polluted areas, and subsequently had normal or elevated POP levels. Clinical parameters related to e.g. hormone levels and liver enzymes were measured as biomarkers for adverse health effects. The Infinium 450K BeadChip was used to assess DNA methylation in a representative subset of 34 men. We identified multiple genes with differentially methylated regions (DMRs; false discovery rate <0.05) related to POP levels. Several of these genes are involved in carcinogenesis (e.g. BRCA1, MAGEE2, HOXA5), the immune system (e.g. RNF39, HLA-DQB1), retinol homeostasis (DHRS4L2), or in metabolism (CYP1A1). The DMRs in these genes show mean methylation differences up to 7.4% when comparing low- and high-exposed men, with a mean difference up to 14.4% for single positions within a DMR. Clinical parameters were not significantly associated with serum POP levels. This is the first explorative study investigating extensive DNA methylation in relation to serum POP levels among men. We observed that elevated POP levels are associated with aberrant DNA methylation profiles in adult men who consumed high-polluted eel. These preliminary findings warrant further confirmation in other populations.

Persistent organic pollutants alter DNA methylation during human adipocyte differentiation.

Ubiquitous persistent organic pollutants (POPs) can accumulate in humans where they might influence differentiation of adipocytes. The aim of this study was to investigate whether DNA methylation is one of the underlying mechanisms by which POPs affect adipocyte differentiation, and to what extent DNA methylation can be related to gene transcription. Adipocyte differentiation was induced in two human cell models with continuous exposure to different POPs throughout differentiation. From the seven tested POPs, perfluorooctanesulfonic acid (PFOS) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decreased lipid accumulation, while tributyltin (TBT) increased lipid accumulation. In human mesenchymal stem cells (hMSCs), TCDD and TBT induced opposite gene expression profiles, whereas after PFOS exposure gene expression remained relatively stable. Genome-wide DNA methylation analysis showed that all three POPs affected DNA methylation patterns in adipogenic and other genes, possibly related to the phenotypic outcome, but without concomitant gene expression changes. Differential methylation was predominantly detected in intergenic regions, where the biological relevance of alterations in DNA methylation is unclear. This study demonstrates that POPs, at environmentally relevant levels, are able to induce differential DNA methylation in human differentiating adipocytes.

Fibroblast growth factor 21 reflects liver fat accumulation and dysregulation of signalling pathways in the liver of C57BL/6J mice.

Fibroblast growth factor 21 (Fgf21) has emerged as a potential plasma marker to diagnose non-alcoholic fatty liver disease (NAFLD). To study the molecular processes underlying the association of plasma Fgf21 with NAFLD, we explored the liver transcriptome data of a mild NAFLD model of aging C57BL/6J mice at 12, 24, and 28 months of age. The plasma Fgf21 level significantly correlated with intrahepatic triglyceride content. At the molecular level, elevated plasma Fgf21 levels were associated with dysregulated metabolic and cancer-related pathways. The up-regulated Fgf21 levels in NAFLD were implied to be a protective response against the NAFLD-induced adverse effects, e.g. lipotoxicity, oxidative stress and endoplasmic reticulum stress. An in vivo PPARα challenge demonstrated the dysregulation of PPARα signalling in the presence of NAFLD, which resulted in a stochastically increasing hepatic expression of Fgf21. Notably, elevated plasma Fgf21 was associated with declining expression of Klb, Fgf21's crucial co-receptor, which suggests a resistance to Fgf21. Therefore, although liver fat accumulation is a benign stage of NAFLD, the elevated plasma Fgf21 likely indicated vulnerability to metabolic stressors that may contribute towards progression to end-stage NAFLD. In conclusion, plasma levels of Fgf21 reflect liver fat accumulation and dysregulation of metabolic pathways in the liver.

Longer lifespan in male mice treated with a weakly estrogenic agonist, an antioxidant, an α-glucosidase inhibitor or a Nrf2-inducer.

The National Institute on Aging Interventions Testing Program (ITP) evaluates agents hypothesized to increase healthy lifespan in genetically heterogeneous mice. Each compound is tested in parallel at three sites, and all results are published. We report the effects of lifelong treatment of mice with four agents not previously tested: Protandim, fish oil, ursodeoxycholic acid (UDCA) and metformin - the latter with and without rapamycin, and two drugs previously examined: 17-α-estradiol and nordihydroguaiaretic acid (NDGA), at doses greater and less than used previously. 17-α-estradiol at a threefold higher dose robustly extended both median and maximal lifespan, but still only in males. The male-specific extension of median lifespan by NDGA was replicated at the original dose, and using doses threefold lower and higher. The effects of NDGA were dose dependent and male specific but without an effect on maximal lifespan. Protandim, a mixture of botanical extracts that activate Nrf2, extended median lifespan in males only. Metformin alone, at a dose of 0.1% in the diet, did not significantly extend lifespan. Metformin (0.1%) combined with rapamycin (14 ppm) robustly extended lifespan, suggestive of an added benefit, based on historical comparison with earlier studies of rapamycin given alone. The α-glucosidase inhibitor, acarbose, at a concentration previously tested (1000 ppm), significantly increased median longevity in males and 90th percentile lifespan in both sexes, even when treatment was started at 16 months. Neither fish oil nor UDCA extended lifespan. These results underscore the reproducibility of ITP longevity studies and illustrate the importance of identifying optimal doses in lifespan studies.

Comprehensive DNA Methylation and Gene Expression Profiling in Differentiating Human Adipocytes.

Insight into the processes controlling adipogenesis is important in the battle against the obesity epidemic and its related disorders. The transcriptional regulatory cascade involved in adipocyte differentiation has been extensively studied, however, the mechanisms driving the transcription activation are still poorly understood. In this study, we explored the involvement of DNA methylation in transcriptional regulation during adipocyte differentiation of primary human mesenchymal stem cells (hMSCs). Genome-wide changes in DNA methylation were measured using the Illumina 450K BeadChip. In addition, expression of 84 adipogenic genes was determined, of which 43 genes showed significant expression changes during the differentiation process. Among these 43 differentially expressed genes, differentially methylated regions (DMRs) were detected in only three genes. By comparing genome-wide DNA methylation profiles in undifferentiated and differentiated adipocytes 793 significant DMRs were detected. Pathway analysis revealed the adipogenesis pathway as the most statistically significant, although only a small number of genes were differentially methylated. Genome-wide DNA methylation changes for single probes were most often located in intergenic regions, and underrepresented close to the transcription start site. In conclusion, DNA methylation remained relatively stable during adipocyte differentiation, implying that changes in DNA methylation are not the underlying mechanism regulating gene expression during adipocyte differentiation. J. Cell. Biochem. 117: 2707-2718, 2016. © 2016 Wiley Periodicals, Inc.

The effects of long-term daily folic acid and vitamin B12 supplementation on genome-wide DNA methylation in elderly subjects.

BACKGROUND: Folate and its synthetic form folic acid function as donor of one-carbon units and have been, together with other B-vitamins, implicated in programming of epigenetic processes such as DNA methylation during early development. To what extent regulation of DNA methylation can be altered via B-vitamins later in life, and how this relates to health and disease, is not exactly known. The aim of this study was to identify effects of long-term supplementation with folic acid and vitamin B12 on genome-wide DNA methylation in elderly subjects. This project was part of a randomized, placebo-controlled trial on effects of supplemental intake of folic acid and vitamin B12 on bone fracture incidence (B-vitamins for the PRevention Of Osteoporotic Fractures (B-PROOF) study). Participants with mildly elevated homocysteine levels, aged 65-75 years, were randomly assigned to take 400 µg folic acid and 500 µg vitamin B12 per day or a placebo during an intervention period of 2 years. DNA was isolated from buffy coats, collected before and after intervention, and genome-wide DNA methylation was determined in 87 participants (n = 44 folic acid/vitamin B12, n = 43 placebo) using the Infinium HumanMethylation450 BeadChip. RESULTS: After intervention with folic acid and vitamin B12, 162 (versus 14 in the placebo group) of the 431,312 positions were differentially methylated as compared to baseline. Comparisons of the DNA methylation changes in the participants receiving folic acid and vitamin B12 versus placebo revealed one single differentially methylated position (cg19380919) with a borderline statistical significance. However, based on the analyses of differentially methylated regions (DMRs) consisting of multiple positions, we identified 6 regions that differed statistically significantly between the intervention and placebo group. Pronounced changes were found for regions in the DIRAS3, ARMC8, and NODAL genes, implicated in carcinogenesis and early embryonic development. Furthermore, serum levels of folate and vitamin B12 or plasma homocysteine were related to DNA methylation of 173, 425, and 11 regions, respectively. Interestingly, for several members of the developmental HOX genes, DNA methylation was related to serum levels of folate. CONCLUSIONS: Long-term supplementation with folic acid and vitamin B12 in elderly subjects resulted in effects on DNA methylation of several genes, among which genes implicated in developmental processes.

Behavioural changes are a major contributing factor in the reduction of sarcopenia in caloric-restricted ageing mice.

BACKGROUND: In rodent models, caloric restriction (CR) with maintenance of adequate micronutrient supply has been reported to increase lifespan and to reduce age-induced muscle loss (sarcopenia) during ageing. In the present study, we further investigated effects of CR on the onset and severity of sarcopenia in ageing male C57BL/6 J mice. The aim of this study was to investigate whether CR induces changes in behaviour of the animals that could contribute to the pronounced health-promoting effects of CR in rodents. In addition, we aimed to investigate in more detail the effects of CR on the onset and severity of sarcopenia. METHODS: The mice received either an ad libitum diet (control) or a diet matching 70 E% of the control diet (C). Daily activity, body composition (dual energy X-ray absorptiometry), grip strength, insulin sensitivity, and general agility and balance were determined at different ages. Mice were killed at 4, 12, 24, and 28 months. Skeletal muscles of the hind limb were dissected, and the muscle extensor digitorum longus muscle was used for force-frequency measurements. The musculus tibialis was used for real-time quantitative PCR analysis. RESULTS: From the age of 12 months, CR animals were nearly half the weight of the control animals, which was mainly related to a lower fat mass. In the control group, the hind limb muscles showed a decline in mass at 24 or 28 months of age, which was not present in the CR group. Moreover, insulin sensitivity (oral glucose tolerance test) was higher in this group and the in vivo and ex vivo grip strength did not differ between the two groups. In the hours before food was provided, CR animals were far more active than control animals, while total daily activity was not increased. Moreover, agility test indicated that CR animals were better climbers and showed more climbing behaviours. CONCLUSIONS: Our study confirms earlier findings that in CR animals less sarcopenia is present. The mice on the CR diet, however, showed specific behavioural changes characterized by higher bursts of activity within a short time frame before consumption of a 70 E% daily meal. We hypothesize that the positive effects of CR on muscle maintenance in rodents are not merely a direct consequence of a lower energy intake but also related to a more active behaviour in a specific time frame. The burst of activity just before immediate start of eating, might lead to a highly effective use of the restricted protein sources available.

Steroid hormone related effects of marine persistent organic pollutants in human H295R adrenocortical carcinoma cells.

Persistent organic pollutants (POPs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorobiphenyl (PCB) 126 and 153, perfluorooctanesulfonic acid (PFOS), hexabromocyclododecane (HBCD), 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), tributyltin (TBT), and methylmercury (MeHg) can be accumulated in seafood and then form a main source for human exposure. Some POPs have been associated with changes in steroid hormone levels in both humans and animals. This study describes the in vitro effects of these POPs and mixtures thereof in H295R adrenocortical carcinoma cells. Relative responses for 13 steroid hormones and 7 genes involved in the steroidogenic pathway, and CYP1A1, were analyzed. PFOS induced the most pronounced effects on steroid hormone levels by significantly affecting 9 out of 13 hormone levels measured, with the largest increases found for 17ß-estradiol, corticosterone, and cortisol. Furthermore, TCDD, both PCBs, and TBT significantly altered steroidogenesis. Increased steroid hormone levels were accompanied by related increased gene expression levels. The differently expressed genes were MC2R, CYP11B1, CYP11B2, and CYP19A1 and changes in gene expression levels were more sensitive than changes in hormone levels. The POP mixtures tested showed mostly additive effects, especially for DHEA and 17ß-estradiol levels. This study shows that some seafood POPs are capable of altering steroidogenesis in H295R cells at concentrations that mixtures might reach in human blood, suggesting that adverse health effects cannot be excluded.

Sexually dimorphic characteristics of the small intestine and colon of prepubescent C57BL/6 mice.

BACKGROUND: There is increasing appreciation for sexually dimorphic effects, but the molecular mechanisms underlying these effects are only partially understood. In the present study, we explored transcriptomics and epigenetic differences in the small intestine and colon of prepubescent male and female mice. In addition, the microbiota composition of the colonic luminal content has been examined. METHODS: At postnatal day 14, male and female C57BL/6 mice were sacrificed and the small intestine, colon and content of luminal colon were isolated. Gene expression of both segments of the intestine was analysed by microarray analysis. DNA methylation of the promoter regions of selected sexually dimorphic genes was examined by pyrosequencing. Composition of the microbiota was explored by deep sequencing. RESULTS: Sexually dimorphic genes were observed in both segments of the intestine of 2-week-old mouse pups, with a stronger effect in the small intestine. Amongst the total of 349 genes displaying a sexually dimorphic effect in the small intestine and/or colon, several candidates exhibited a previously established function in the intestine (i.e. Nts, Nucb2, Alox5ap and Retnlγ). In addition, differential expression of genes linked to intestinal bowel disease (i.e. Ccr3, Ccl11 and Tnfr) and colorectal cancer development (i.e. Wt1 and Mmp25) was observed between males and females. Amongst the genes displaying significant sexually dimorphic expression, nine genes were histone-modifying enzymes, suggesting that epigenetic mechanisms might be a potential underlying regulatory mechanism. However, our results reveal no significant changes in DNA methylation of analysed CpGs within the selected differentially expressed genes. With respect to the bacterial community composition in the colon, a dominant effect of litter origin was found but no significant sex effect was detected. However, a sex effect on the dominance of specific taxa was observed. CONCLUSIONS: This study reveals molecular dissimilarities between males and females in the small intestine and colon of prepubescent mice, which might underlie differences in physiological functioning and in disease predisposition in the two sexes.

Genome-wide age-related changes in DNA methylation and gene expression in human PBMCs.

Aging is a progressive process that results in the accumulation of intra- and extracellular alterations that in turn contribute to a reduction in health. Age-related changes in DNA methylation have been reported before and may be responsible for aging-induced changes in gene expression, although a causal relationship has yet to be shown. Using genome-wide assays, we analyzed age-induced changes in DNA methylation and their effect on gene expression with and without transient induction with the synthetic transcription modulating agent WY14,643. To demonstrate feasibility of the approach, we isolated peripheral blood mononucleated cells (PBMCs) from five young and five old healthy male volunteers and cultured them with or without WY14,643. Infinium 450K BeadChip and Affymetrix Human Gene 1.1 ST expression array analysis revealed significant differential methylation of at least 5 % (ΔYO > 5 %) at 10,625 CpG sites between young and old subjects, but only a subset of the associated genes were also differentially expressed. Age-related differential methylation of previously reported epigenetic biomarkers of aging including ELOVL2, FHL2, PENK, and KLF14 was confirmed in our study, but these genes did not display an age-related change in gene expression in PBMCs. Bioinformatic analysis revealed that differentially methylated genes that lack an age-related expression change predominantly represent genes involved in carcinogenesis and developmental processes, and expression of most of these genes were silenced in PBMCs. No changes in DNA methylation were found in genes displaying transiently induced changes in gene expression. In conclusion, aging-induced differential methylation often targets developmental genes and occurs mostly without change in gene expression.

Associations of 25-hydroxyvitamin D with fasting glucose, fasting insulin, dementia and depression in European elderly: the SENECA study.

PURPOSE: The classical consequence of vitamin D deficiency is osteomalacia, but recent insights into the function of vitamin D suggest that it may play a role in other body systems as well. The objective of this study was to examine the association between 25-hydroxyvitamin D (25(OH)D) and markers of glucose metabolism (n = 593), global cognitive functioning (n = 116) and depression (n = 118) in European elderly participating in the SENECA study. Moreover, we wanted to explore whether the observed associations of 25(OH)D with depression and global cognitive performance were mediated by fasting plasma glucose (FPG) levels. METHODS: Cross-sectional associations between 25(OH)D and FPG, fasting plasma insulin (FPI) and homeostatic model assessment-insulin resistance (HOMA-IR), a marker of insulin resistance, were estimated from multiple regression analyses. Associations of 25(OH)D with global cognitive functioning (Mini Mental State Examination) and depression (Geriatric Depression Scale) were examined using Poisson regression. RESULTS: An inverse association was observed between 25(OH)D and FPG (ß-0.001), indicating a 1 % decrease in FPG per 10 nmol/L increase in 25(OH)D, but after full adjustment for demographic factors, lifestyle factors and calcium intake, this association was not statistically significant (P = 0.07). Although participants with intermediate and high serum 25(OH)D levels showed a tendency towards a lower depression score after adjustment for demographic and lifestyle factors, RR and 95 % CI: 0.73 (0.51-1.04) and 0.76 (0.52-1.11), respectively, these findings were not statistically significant. CONCLUSION: An inverse association of 25(OH)D with depression and FPG was observed, but this association was not statistically significant. There was no association between 25(OH)D with FPI and HOMA-IR or with global cognitive functioning. More studies are needed to further explore the possible role of vitamin D in the various body systems.

Inhibition of methylation decreases osteoblast differentiation via a non-DNA-dependent methylation mechanism.

S-adenosylmethionine (SAM)-dependent methylation of biological molecules including DNA and proteins is rapidly being uncovered as a critical mechanism for regulation of cellular processes. We investigated the effects of reduced SAM-dependent methylation on osteoblast differentiation by using periodate oxidized adenosine (ADOX), an inhibitor of SAM-dependent methyltransferases. The capacity of this agent to modulate osteoblast differentiation was analyzed under non-osteogenic control conditions and during growth factor-induced differentiation and compared with the effect of inhibition of DNA methylation by 5-Aza-2'-deoxycytidine (5-Aza-CdR). Without applying specific osteogenic triggers, both ADOX and 5-Aza-CdR induced mRNA expression of the osteoblast markers Alp, Osx, and Ocn in murine C2C12 cells. Under osteogenic conditions, ADOX inhibited differentiation of both human mesenchymal stem cells and C2C12 cells. Gene expression analysis of early (Msx2, Dlx5, Runx2) and late (Alp, Osx, Ocn) osteoblast markers during bone morphogenetic protein 2-induced C2C12 osteoblast differentiation revealed that ADOX only reduced expression of the late phase Runx2 target genes. By using a Runx2-responsive luciferase reporter (6xOSE), we showed that ADOX reduced the activity of Runx2, while 5-Aza-CdR had no effect. Taken together, our data suggest that decreased SAM-dependent methyltransferase activity leads to impaired osteoblast differentiation via non-DNA-dependent methylation mechanisms and that methylation is a regulator of Runx2-controlled gene expression.

Microarray analysis of bone morphogenetic protein, transforming growth factor beta, and activin early response genes during osteoblastic cell differentiation.

Bone morphogenetic protein (BMP) 2, a member of the transforming growth factor (TGF) beta family, is a potent regulator of osteoblast differentiation. In addition, both TGF-beta and activin A can either induce bone formation or inhibit bone formation depending on cell type and differentiation status. Although much is known about the receptors and intracellular second messengers involved in the action of TGF-beta family members, little is known about how selectivity in the biological response of individual family members is controlled. In this study, we have investigated selective gene induction by BMP-2, TGF-beta1 and activin A in relation to their ability to control differentiation of mouse mesenchymal precursor cells C2C12 into osteoblastic cells. TGF-beta1 can inhibit BMP-2-induced differentiation of these cells, whereas activin A was found to be without morphogenetic effect. Using a gene expression microarray approach covering 8636 sequences, we have identified a total of 57 established genes and expressed sequence tags (ESTs) that were either up-regulated or down-regulated 2 h after treatment with at least one of these three stimuli. With respect to the established genes, 15 new target genes for TGF-beta family members thus were identified. Furthermore, a set of transcripts was identified, which was oppositely regulated by TGF-beta1 and BMP-2. Based on the inverse biological effects of TGF-beta1 and BMP-2 on C2C12 cells, these genes are important candidates for controlling the process of growth factor-induced osteoblast differentiation.