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Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors.

Schroeder TM, Nair AK, Staggs R, Lamblin AF, Westendorf JJ - BMC Genomics (2007)

Bottom Line: We also identified eight growth factors and growth factor receptor genes that are significantly altered by each of the HDIs, including Frizzled related proteins 1 and 4, which modulate the Wnt signaling pathway.Similarly, the regulation of Wnt receptor genes indicates that this crucial pathway in osteoblast development is also affected by HDIs.These data support the hypothesis that HDIs regulate the expression of genes that promote osteoblast differentiation and maturation.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Cancer Center, and Department of Orthopaedic Surgery, University of Minnesota, MMC 806, 420 Delaware Street SW, Minneapolis, MN, USA. tania.schroeder@ammd.com

ABSTRACT

Background: Osteoblast differentiation requires the coordinated stepwise expression of multiple genes. Histone deacetylase inhibitors (HDIs) accelerate the osteoblast differentiation process by blocking the activity of histone deacetylases (HDACs), which alter gene expression by modifying chromatin structure. We previously demonstrated that HDIs and HDAC3 shRNAs accelerate matrix mineralization and the expression of osteoblast maturation genes (e.g. alkaline phosphatase, osteocalcin). Identifying other genes that are differentially regulated by HDIs might identify new pathways that contribute to osteoblast differentiation.

Results: To identify other osteoblast genes that are altered early by HDIs, we incubated MC3T3-E1 preosteoblasts with HDIs (trichostatin A, MS-275, or valproic acid) for 18 hours in osteogenic conditions. The promotion of osteoblast differentiation by HDIs in this experiment was confirmed by osteogenic assays. Gene expression profiles relative to vehicle-treated cells were assessed by microarray analysis with Affymetrix GeneChip 430 2.0 arrays. The regulation of several genes by HDIs in MC3T3-E1 cells and primary osteoblasts was verified by quantitative real-time PCR. Nine genes were differentially regulated by at least two-fold after exposure to each of the three HDIs and six were verified by PCR in osteoblasts. Four of the verified genes (solute carrier family 9 isoform 3 regulator 1 (Slc9a3r1), sorbitol dehydrogenase 1, a kinase anchor protein, and glutathione S-transferase alpha 4) were induced. Two genes (proteasome subunit, beta type 10 and adaptor-related protein complex AP-4 sigma 1) were suppressed. We also identified eight growth factors and growth factor receptor genes that are significantly altered by each of the HDIs, including Frizzled related proteins 1 and 4, which modulate the Wnt signaling pathway.

Conclusion: This study identifies osteoblast genes that are regulated early by HDIs and indicates pathways that might promote osteoblast maturation following HDI exposure. One gene whose upregulation following HDI treatment is consistent with this notion is Slc9a3r1. Also known as NHERF1, Slc9a3r1 is required for optimal bone density. Similarly, the regulation of Wnt receptor genes indicates that this crucial pathway in osteoblast development is also affected by HDIs. These data support the hypothesis that HDIs regulate the expression of genes that promote osteoblast differentiation and maturation.

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HDI treatment accelerates the appearance of alkaline phosphatase activity in differentiating MC3T3 cells. MC3T3 cells were cultured for the indicated times in osteogenic medium. The medium was changed three days with the HDIs or vehicle added only at day 0. Fold change in ALP activity is shown in relation to values obtained at the start of the culture (day 0). * denotes a statistically significant change of p < 0.01 by one-way ANOVA of the HDI-treated sample versus the DMSO-treated sample at that time point.
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Figure 1: HDI treatment accelerates the appearance of alkaline phosphatase activity in differentiating MC3T3 cells. MC3T3 cells were cultured for the indicated times in osteogenic medium. The medium was changed three days with the HDIs or vehicle added only at day 0. Fold change in ALP activity is shown in relation to values obtained at the start of the culture (day 0). * denotes a statistically significant change of p < 0.01 by one-way ANOVA of the HDI-treated sample versus the DMSO-treated sample at that time point.

Mentions: We previously demonstrated that HDIs accelerate and enhance alkaline phosphatase expression by MC3T3-E1 pre-osteoblasts after three days. By the second week, the HDI-exposed cultures expressed higher levels of genes associated with osteoblast differentiation (e.g. bone siaoloprotein, osteopontin and osteocalcin) than control cells [18]. The goal of this study was to identify genes that are affected early (within the first 18 hours) by several HDIs because they are more likely to be initiators or early regulators of the process. To demonstrate the differentiation potential of the HDI and vehicle-treated cells used in the microarray experiment, cells in parallel cultures were allowed to differentiate for up to seven days. During the differentiation process, lysates were taken at days 0, 1, 4, and 7 and alkaline phosphatase activity was measured. In the DMSO-treated cells, alkaline phosphatase activity increased steadily over time demonstrating that the cells were differentiating appropriately (Figure 1). Alkaline phosphatase activity also increased in the HDI-treated cells and was generally higher in HDI-exposed cells relative to vehicle-treated cells at days 4 and 7 (Figure 1). These results demonstrate that the MC3T3 cells used for microarray analysis differentiated appropriately and that the HDIs accelerated differentiation as expected from our previous studies [18].


Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors.

Schroeder TM, Nair AK, Staggs R, Lamblin AF, Westendorf JJ - BMC Genomics (2007)

HDI treatment accelerates the appearance of alkaline phosphatase activity in differentiating MC3T3 cells. MC3T3 cells were cultured for the indicated times in osteogenic medium. The medium was changed three days with the HDIs or vehicle added only at day 0. Fold change in ALP activity is shown in relation to values obtained at the start of the culture (day 0). * denotes a statistically significant change of p < 0.01 by one-way ANOVA of the HDI-treated sample versus the DMSO-treated sample at that time point.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2147034&req=5

Figure 1: HDI treatment accelerates the appearance of alkaline phosphatase activity in differentiating MC3T3 cells. MC3T3 cells were cultured for the indicated times in osteogenic medium. The medium was changed three days with the HDIs or vehicle added only at day 0. Fold change in ALP activity is shown in relation to values obtained at the start of the culture (day 0). * denotes a statistically significant change of p < 0.01 by one-way ANOVA of the HDI-treated sample versus the DMSO-treated sample at that time point.
Mentions: We previously demonstrated that HDIs accelerate and enhance alkaline phosphatase expression by MC3T3-E1 pre-osteoblasts after three days. By the second week, the HDI-exposed cultures expressed higher levels of genes associated with osteoblast differentiation (e.g. bone siaoloprotein, osteopontin and osteocalcin) than control cells [18]. The goal of this study was to identify genes that are affected early (within the first 18 hours) by several HDIs because they are more likely to be initiators or early regulators of the process. To demonstrate the differentiation potential of the HDI and vehicle-treated cells used in the microarray experiment, cells in parallel cultures were allowed to differentiate for up to seven days. During the differentiation process, lysates were taken at days 0, 1, 4, and 7 and alkaline phosphatase activity was measured. In the DMSO-treated cells, alkaline phosphatase activity increased steadily over time demonstrating that the cells were differentiating appropriately (Figure 1). Alkaline phosphatase activity also increased in the HDI-treated cells and was generally higher in HDI-exposed cells relative to vehicle-treated cells at days 4 and 7 (Figure 1). These results demonstrate that the MC3T3 cells used for microarray analysis differentiated appropriately and that the HDIs accelerated differentiation as expected from our previous studies [18].

Bottom Line: We also identified eight growth factors and growth factor receptor genes that are significantly altered by each of the HDIs, including Frizzled related proteins 1 and 4, which modulate the Wnt signaling pathway.Similarly, the regulation of Wnt receptor genes indicates that this crucial pathway in osteoblast development is also affected by HDIs.These data support the hypothesis that HDIs regulate the expression of genes that promote osteoblast differentiation and maturation.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Cancer Center, and Department of Orthopaedic Surgery, University of Minnesota, MMC 806, 420 Delaware Street SW, Minneapolis, MN, USA. tania.schroeder@ammd.com

ABSTRACT

Background: Osteoblast differentiation requires the coordinated stepwise expression of multiple genes. Histone deacetylase inhibitors (HDIs) accelerate the osteoblast differentiation process by blocking the activity of histone deacetylases (HDACs), which alter gene expression by modifying chromatin structure. We previously demonstrated that HDIs and HDAC3 shRNAs accelerate matrix mineralization and the expression of osteoblast maturation genes (e.g. alkaline phosphatase, osteocalcin). Identifying other genes that are differentially regulated by HDIs might identify new pathways that contribute to osteoblast differentiation.

Results: To identify other osteoblast genes that are altered early by HDIs, we incubated MC3T3-E1 preosteoblasts with HDIs (trichostatin A, MS-275, or valproic acid) for 18 hours in osteogenic conditions. The promotion of osteoblast differentiation by HDIs in this experiment was confirmed by osteogenic assays. Gene expression profiles relative to vehicle-treated cells were assessed by microarray analysis with Affymetrix GeneChip 430 2.0 arrays. The regulation of several genes by HDIs in MC3T3-E1 cells and primary osteoblasts was verified by quantitative real-time PCR. Nine genes were differentially regulated by at least two-fold after exposure to each of the three HDIs and six were verified by PCR in osteoblasts. Four of the verified genes (solute carrier family 9 isoform 3 regulator 1 (Slc9a3r1), sorbitol dehydrogenase 1, a kinase anchor protein, and glutathione S-transferase alpha 4) were induced. Two genes (proteasome subunit, beta type 10 and adaptor-related protein complex AP-4 sigma 1) were suppressed. We also identified eight growth factors and growth factor receptor genes that are significantly altered by each of the HDIs, including Frizzled related proteins 1 and 4, which modulate the Wnt signaling pathway.

Conclusion: This study identifies osteoblast genes that are regulated early by HDIs and indicates pathways that might promote osteoblast maturation following HDI exposure. One gene whose upregulation following HDI treatment is consistent with this notion is Slc9a3r1. Also known as NHERF1, Slc9a3r1 is required for optimal bone density. Similarly, the regulation of Wnt receptor genes indicates that this crucial pathway in osteoblast development is also affected by HDIs. These data support the hypothesis that HDIs regulate the expression of genes that promote osteoblast differentiation and maturation.

Show MeSH
Related in: MedlinePlus