Limits...
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.

Show MeSH

Related in: MedlinePlus

Verification of Psmb10 and Ap4s1 as HDI-suppressed genes. Primary calvarial osteoblasts were cultured in osteogenic medium containing the indicated HDI or DMSO. mRNAs were isolated after 18 hours and subjected to quantitative real-time PCR with primers for Psmb10 (A) or Ap4s1 (B). Values are relative to those obtained from DMSO-treated samples at each time point and represent the mean of three samples. ** denotes p < 0.05 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
getmorefigures.php?uid=PMC2147034&req=5

Figure 5: Verification of Psmb10 and Ap4s1 as HDI-suppressed genes. Primary calvarial osteoblasts were cultured in osteogenic medium containing the indicated HDI or DMSO. mRNAs were isolated after 18 hours and subjected to quantitative real-time PCR with primers for Psmb10 (A) or Ap4s1 (B). Values are relative to those obtained from DMSO-treated samples at each time point and represent the mean of three samples. ** denotes p < 0.05 by one-way ANOVA of the HDI-treated sample versus the DMSO-treated sample at that time point.

Mentions: The temporal regulation of several identified genes was determined by quantitative real-time PCRs using mRNAs isolated from independent cultures of MC3T3 cells or primary murine calvarial osteoblasts. Consistent with the GeneChip analysis, TSA was a more potent inducer of Slc9a3r1 than MS-275 or VPA in both MC3T3 cells (Figure 4A) and primary osteoblasts (Figure 4B). Slc9a3r1 induction was detectable at both the RNA and protein levels as early as 4 to 6 hours after HDI exposure (Figure 4B and 4E). Similar effects were observed with Sdh1 mRNA in both MC3T3 (Figure 4C) and primary osteoblasts (Figure 4D) except VPA induced a more rapid increase in Sdh1 expression than TSA. The induction of Akap12 was also confirmed in primary osteoblasts (data not shown). The suppression of Psmb10 and Ap4s1 was confirmed in primary osteoblasts (Figure 5).


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)

Verification of Psmb10 and Ap4s1 as HDI-suppressed genes. Primary calvarial osteoblasts were cultured in osteogenic medium containing the indicated HDI or DMSO. mRNAs were isolated after 18 hours and subjected to quantitative real-time PCR with primers for Psmb10 (A) or Ap4s1 (B). Values are relative to those obtained from DMSO-treated samples at each time point and represent the mean of three samples. ** denotes p < 0.05 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 5: Verification of Psmb10 and Ap4s1 as HDI-suppressed genes. Primary calvarial osteoblasts were cultured in osteogenic medium containing the indicated HDI or DMSO. mRNAs were isolated after 18 hours and subjected to quantitative real-time PCR with primers for Psmb10 (A) or Ap4s1 (B). Values are relative to those obtained from DMSO-treated samples at each time point and represent the mean of three samples. ** denotes p < 0.05 by one-way ANOVA of the HDI-treated sample versus the DMSO-treated sample at that time point.
Mentions: The temporal regulation of several identified genes was determined by quantitative real-time PCRs using mRNAs isolated from independent cultures of MC3T3 cells or primary murine calvarial osteoblasts. Consistent with the GeneChip analysis, TSA was a more potent inducer of Slc9a3r1 than MS-275 or VPA in both MC3T3 cells (Figure 4A) and primary osteoblasts (Figure 4B). Slc9a3r1 induction was detectable at both the RNA and protein levels as early as 4 to 6 hours after HDI exposure (Figure 4B and 4E). Similar effects were observed with Sdh1 mRNA in both MC3T3 (Figure 4C) and primary osteoblasts (Figure 4D) except VPA induced a more rapid increase in Sdh1 expression than TSA. The induction of Akap12 was also confirmed in primary osteoblasts (data not shown). The suppression of Psmb10 and Ap4s1 was confirmed in primary osteoblasts (Figure 5).

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