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Effects of Age and Estrogen on Skeletal Gene Expression in Humans as Assessed by RNA Sequencing.

Farr JN, Roforth MM, Fujita K, Nicks KM, Cunningham JM, Atkinson EJ, Therneau TM, McCready LK, Peterson JM, Drake MT, Monroe DG, Khosla S - PLoS ONE (2015)

Bottom Line: Interestingly, the LEF1 transcription factor, which is a classical downstream target of the Wnt/β-catenin signaling pathway, was significantly downregulated in the bones from the old versus young women; consistent with this, LEF1 binding sites were significantly enriched in the promoter regions of the differentially expressed genes in the old versus young women, suggesting that aging was associated with alterations in Wnt signaling in bone.In conclusion, our data demonstrate that aging alters a substantial portion of the skeletal transcriptome, whereas E therapy appears to have significant, albeit less wide-ranging effects.These data provide a valuable resource for the potential identification of novel biomarkers associated with age-related bone loss and also highlight potential pathways that could be targeted to treat osteoporosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology, Department of Medicine, Mayo Clinic College of Medicine, Rochester, MN, 55905, United States of America; Robert and Arlene Kogod Center on Aging, Mayo Clinic College of Medicine, Rochester, MN, 55905, United States of America.

ABSTRACT

Unlabelled: Precise delineation of the specific genes and pathways altered with aging and estrogen (E) therapy may lead to new skeletal biomarkers and the development of novel bone therapeutics. Previous human bone studies, however, have been limited by only examining pre-specified genes and pathways. High-throughput RNA sequencing (RNAseq), on the other hand, offers an unbiased approach to examine the entire transcriptome. Here we present an RNAseq analysis of human bone samples, obtained from iliac crest needle biopsies, to yield the first in vivo interrogation of all genes and pathways that may be altered in bone with aging and E therapy in humans. 58 healthy women were studied, including 19 young women (mean age ± SD, 30.3 ± 5.4 years), 19 old women (73.1 ± 6.6 years), and 20 old women treated with 3 weeks of E therapy (70.5 ± 5.2 years). Using generally accepted criteria (false discovery rate [q] < 0.10), aging altered a total of 678 genes and 12 pathways, including a subset known to regulate bone metabolism (e.g., Notch). Interestingly, the LEF1 transcription factor, which is a classical downstream target of the Wnt/β-catenin signaling pathway, was significantly downregulated in the bones from the old versus young women; consistent with this, LEF1 binding sites were significantly enriched in the promoter regions of the differentially expressed genes in the old versus young women, suggesting that aging was associated with alterations in Wnt signaling in bone. Further, of the 21 unique genes altered in bone by E therapy, the expression of INHBB (encoding for the inhibin, beta B polypeptide), which decreased with aging (by 0.6-fold), was restored to young adult levels in response to E therapy. In conclusion, our data demonstrate that aging alters a substantial portion of the skeletal transcriptome, whereas E therapy appears to have significant, albeit less wide-ranging effects. These data provide a valuable resource for the potential identification of novel biomarkers associated with age-related bone loss and also highlight potential pathways that could be targeted to treat osteoporosis.

Trial registration: ClinicalTrials.gov NCT02349113.

No MeSH data available.


Related in: MedlinePlus

Effects of age and estrogen (E) on serum sclerostin levels.Serum sclerostin levels in the young versus old subjects (A) and in the old versus E-treated subjects (B) by the Biomedica and Meso Scale Discovery (MSD) assays. Data are mean ± SEM; note the difference in scales for the two sclerostin assays. *p < 0.05; †p < 0.01.
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pone.0138347.g004: Effects of age and estrogen (E) on serum sclerostin levels.Serum sclerostin levels in the young versus old subjects (A) and in the old versus E-treated subjects (B) by the Biomedica and Meso Scale Discovery (MSD) assays. Data are mean ± SEM; note the difference in scales for the two sclerostin assays. *p < 0.05; †p < 0.01.

Mentions: Since we have previously reported age [6] and E [7] effects on sclerostin (SOST) mRNA levels by QPCR in these women, we next examined SOST gene expression levels in the RNAseq dataset, as well as serum levels of sclerostin in the young, old, and E-treated subjects using two different sclerostin assays. Consistent with our previous QPCR data [6], [7], there was no effect of age on SOST mRNA levels by RNAseq (fold change in the old versus young = 0.99, p = 0.981), whereas E treatment did significantly reduce SOST mRNA levels (fold change in the E-treated versus untreated old women = 0.59, p = 0.026), although this change did not meet FDR criteria (q = 0.46). Interestingly, although serum sclerostin levels were significantly higher in the old versus young subjects using the Biomedica assay, they were no different in the old versus young subjects using the MSD assay (Fig 4A). By contrast, consistent with both the QPCR and RNAseq data, serum sclerostin levels using both assays were reduced in old women by E therapy (Fig 4B). Thus, the MSD assay more accurately reflected changes in bone SOST mRNA levels with aging and E treatment, showing concordant changes in circulating sclerostin levels under both conditions.


Effects of Age and Estrogen on Skeletal Gene Expression in Humans as Assessed by RNA Sequencing.

Farr JN, Roforth MM, Fujita K, Nicks KM, Cunningham JM, Atkinson EJ, Therneau TM, McCready LK, Peterson JM, Drake MT, Monroe DG, Khosla S - PLoS ONE (2015)

Effects of age and estrogen (E) on serum sclerostin levels.Serum sclerostin levels in the young versus old subjects (A) and in the old versus E-treated subjects (B) by the Biomedica and Meso Scale Discovery (MSD) assays. Data are mean ± SEM; note the difference in scales for the two sclerostin assays. *p < 0.05; †p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581624&req=5

pone.0138347.g004: Effects of age and estrogen (E) on serum sclerostin levels.Serum sclerostin levels in the young versus old subjects (A) and in the old versus E-treated subjects (B) by the Biomedica and Meso Scale Discovery (MSD) assays. Data are mean ± SEM; note the difference in scales for the two sclerostin assays. *p < 0.05; †p < 0.01.
Mentions: Since we have previously reported age [6] and E [7] effects on sclerostin (SOST) mRNA levels by QPCR in these women, we next examined SOST gene expression levels in the RNAseq dataset, as well as serum levels of sclerostin in the young, old, and E-treated subjects using two different sclerostin assays. Consistent with our previous QPCR data [6], [7], there was no effect of age on SOST mRNA levels by RNAseq (fold change in the old versus young = 0.99, p = 0.981), whereas E treatment did significantly reduce SOST mRNA levels (fold change in the E-treated versus untreated old women = 0.59, p = 0.026), although this change did not meet FDR criteria (q = 0.46). Interestingly, although serum sclerostin levels were significantly higher in the old versus young subjects using the Biomedica assay, they were no different in the old versus young subjects using the MSD assay (Fig 4A). By contrast, consistent with both the QPCR and RNAseq data, serum sclerostin levels using both assays were reduced in old women by E therapy (Fig 4B). Thus, the MSD assay more accurately reflected changes in bone SOST mRNA levels with aging and E treatment, showing concordant changes in circulating sclerostin levels under both conditions.

Bottom Line: Interestingly, the LEF1 transcription factor, which is a classical downstream target of the Wnt/β-catenin signaling pathway, was significantly downregulated in the bones from the old versus young women; consistent with this, LEF1 binding sites were significantly enriched in the promoter regions of the differentially expressed genes in the old versus young women, suggesting that aging was associated with alterations in Wnt signaling in bone.In conclusion, our data demonstrate that aging alters a substantial portion of the skeletal transcriptome, whereas E therapy appears to have significant, albeit less wide-ranging effects.These data provide a valuable resource for the potential identification of novel biomarkers associated with age-related bone loss and also highlight potential pathways that could be targeted to treat osteoporosis.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology, Department of Medicine, Mayo Clinic College of Medicine, Rochester, MN, 55905, United States of America; Robert and Arlene Kogod Center on Aging, Mayo Clinic College of Medicine, Rochester, MN, 55905, United States of America.

ABSTRACT

Unlabelled: Precise delineation of the specific genes and pathways altered with aging and estrogen (E) therapy may lead to new skeletal biomarkers and the development of novel bone therapeutics. Previous human bone studies, however, have been limited by only examining pre-specified genes and pathways. High-throughput RNA sequencing (RNAseq), on the other hand, offers an unbiased approach to examine the entire transcriptome. Here we present an RNAseq analysis of human bone samples, obtained from iliac crest needle biopsies, to yield the first in vivo interrogation of all genes and pathways that may be altered in bone with aging and E therapy in humans. 58 healthy women were studied, including 19 young women (mean age ± SD, 30.3 ± 5.4 years), 19 old women (73.1 ± 6.6 years), and 20 old women treated with 3 weeks of E therapy (70.5 ± 5.2 years). Using generally accepted criteria (false discovery rate [q] < 0.10), aging altered a total of 678 genes and 12 pathways, including a subset known to regulate bone metabolism (e.g., Notch). Interestingly, the LEF1 transcription factor, which is a classical downstream target of the Wnt/β-catenin signaling pathway, was significantly downregulated in the bones from the old versus young women; consistent with this, LEF1 binding sites were significantly enriched in the promoter regions of the differentially expressed genes in the old versus young women, suggesting that aging was associated with alterations in Wnt signaling in bone. Further, of the 21 unique genes altered in bone by E therapy, the expression of INHBB (encoding for the inhibin, beta B polypeptide), which decreased with aging (by 0.6-fold), was restored to young adult levels in response to E therapy. In conclusion, our data demonstrate that aging alters a substantial portion of the skeletal transcriptome, whereas E therapy appears to have significant, albeit less wide-ranging effects. These data provide a valuable resource for the potential identification of novel biomarkers associated with age-related bone loss and also highlight potential pathways that could be targeted to treat osteoporosis.

Trial registration: ClinicalTrials.gov NCT02349113.

No MeSH data available.


Related in: MedlinePlus