Mechanical-Stress-Related Epigenetic Regulation of ZIC1 Transcription Factor in the Etiology of Postmenopausal Osteoporosis.

Mechanical loading exerts a profound influence on bone density and architecture, but the exact mechanism is unknown. Our study shows that expression of the neurological transcriptional factor zinc finger of the cerebellum 1 (ZIC1) is markedly increased in trabecular bone biopsies in the lumbar spine compared with the iliac crest, skeletal sites of high and low mechanical stress, respectively. Human trabecular bone transcriptome analyses revealed a strong association between ZIC1 mRNA levels and gene transcripts characteristically associated with osteoblasts, osteocytes and osteoclasts. This supposition is supported by higher ZIC1 expression in iliac bone biopsies from postmenopausal women with osteoporosis compared with age-matched control subjects, as well as strongly significant inverse correlation between ZIC1 mRNA levels and BMI-adjusted bone mineral density (BMD) (Z-score). ZIC1 promoter methylation was decreased in mechanically loaded vertebral bone compared to unloaded normal iliac bone, and its mRNA levels correlated inversely with ZIC1 promoter methylation, thus linking mechanical stress to epigenetic control of gene expression. The findings were corroborated in cultures of rat osteoblast progenitors and osteoblast-like cells. This study demonstrates for the first time how skeletal epigenetic changes that are affected by mechanical forces give rise to marked alteration in bone cell transcriptional activity and translate to human bone pathophysiology.

Heavy-load exercise in older adults activates vasculogenesis and has a stronger impact on muscle gene expression than in young adults.

BACKGROUND: A striking effect of old age is the involuntary loss of muscle mass and strength leading to sarcopenia and reduced physiological functions. However, effects of heavy-load exercise in older adults on diseases and functions as predicted by changes in muscle gene expression have been inadequately studied. METHODS: Thigh muscle global transcriptional activity (transcriptome) was analyzed in cohorts of older and younger adults before and after 12-13 weeks heavy-load strength exercise using Affymetrix microarrays. Three age groups, similarly trained, were compared: younger adults (age 24 ± 4 years), older adults of average age 70 years (Oslo cohort) and above 80 years (old BSU cohort). To increase statistical strength, one of the older cohorts was used for validation. Ingenuity Pathway analysis (IPA) was used to identify predicted biological effects of a gene set that changed expression after exercise, and Principal Component Analysis (PCA) was used to visualize differences in muscle gene expressen between cohorts and individual participants as well as overall changes upon exercise. RESULTS: Younger adults, showed few transcriptome changes, but a marked, significant impact was observed in persons of average age 70 years and even more so in persons above 80 years. The 249 transcripts positively or negatively altered in both cohorts of older adults (q-value < 0.1) were submitted to gene set enrichment analysis using IPA. The transcripts predicted increase in several aspects of "vascularization and muscle contractions", whereas functions associated with negative health effects were reduced, e.g., "Glucose metabolism disorder" and "Disorder of blood pressure". Several genes that changed expression after intervention were confirmed at the genome level by containing single nucleotide variants associated with handgrip strength and muscle expression levels, e.g., CYP4B1 (p = 9.2E-20), NOTCH4 (p = 9.7E-8), and FZD4 (p = 5.3E-7). PCA of the 249 genes indicated a differential pattern of muscle gene expression in young and elderly. However, after exercise the expression patterns in both young and old BSU cohorts were changed in the same direction for the vast majority of participants. CONCLUSIONS: The positive impact of heavy-load strength training on the transcriptome increased markedly with age. The identified molecular changes translate to improved vascularization and muscular strength, suggesting highly beneficial health effects for older adults.

Zic1 transcription factor in bone: neural developmental protein regulates mechanotransduction in osteocytes.

A transcriptome analysis compared gene expression in human bone biopsy samples taken from lumbar spine and iliac crest, sites that experience high and low levels of mechanical stress, respectively. The analysis revealed that the zinc finger protein of cerebellum (Zic) family member transcription factor Zic1 was the most up-regulated gene in the lumbar spine (202-fold; P<10(-7)) in comparison with the iliac crest. Software analysis of differential gene expression in the biopsy samples identified the ciliary-related proteins PATCH1 and GLI-Kruppel family members Gli1 and Gli3 as part of a potential molecular network associated with Zic1. RT-PCR confirmed the expression of Zic1, Gli1, and Gli3 and other related key signaling mediators in osteoblastic cells and osteocytes in vitro. Zic1 was immunolocalized in the cytosol and nucleus of the murine osteocyte cell line MLO-Y4 and osteoblast-like cells MC3T3-E1 and in primary rat osteoblasts. MLO-Y4 cells subjected to prolonged oscillatory fluid flow showed increased localization of Zic1 in the nucleus with diminished levels in the cytosol, but no such changes were seen in MC3T3-E1 cells. A shear stress-induced increase in T-cell factor/lymphoid enhancer factor transcriptional activity was abolished by Zic1 gene silencing. These results suggest that Zic1, perhaps together with Gli1 and Gli3, may act as a link between mechanosensing and Wnt signaling. We conclude that Zic1, a neural developmental transcription factor, plays an important role in shear flow mechanotransduction in osteocytes.

Distinct Subsets of Noncoding RNAs Are Strongly Associated With BMD and Fracture, Studied in Weight-Bearing and Non-Weight-Bearing Human Bone.

We investigated mechanisms resulting in low bone mineral density (BMD) and susceptibility to fracture by comparing noncoding RNAs (ncRNAs) in biopsies of non-weight-bearing (NWB) iliac (n = 84) and weight bearing (WB) femoral (n = 18) postmenopausal bone across BMDs varying from normal (T-score > -1.0) to osteoporotic (T-score ≤ -2.5). Global bone ncRNA concentrations were determined by PCR and microchip analyses. Association with BMD or fracture, adjusted by age and body mass index, were calculated using linear and logistic regression and least absolute shrinkage and selection operator (Lasso) analysis. At 10% false discovery rate (FDR), 75 iliac bone ncRNAs and 94 femoral bone ncRNAs were associated with total hip BMD. Eight of the ncRNAs were common for the two sites, but five of them (miR-484, miR-328-3p, miR-27a-5p, miR-28-3p, and miR-409-3p) correlated positively to BMD in femoral bone, but negatively in iliac bone. Of predicted pathways recognized in bone metabolism, ECM-receptor interaction and proteoglycans in cancer emerged at both sites, whereas fatty acid metabolism and focal adhesion were only identified in iliac bone. Lasso analysis and cross-validations identified sets of nine bone ncRNAs correlating strongly with adjusted total hip BMD in both femoral and iliac bone. Twenty-eight iliac ncRNAs were associated with risk of fracture (FDR < 0.1). The small nucleolar RNAs, RNU44 and RNU48, have a function in stabilization of ribosomal RNAs (rRNAs), and their association with fracture and BMD suggest that aberrant processing of rRNAs may be involved in development of osteoporosis. Cis-eQTL (expressed quantitative trait loci) analysis of the iliac bone biopsies identified two loci associated with microRNAs (miRNAs), one previously identified in a heel-BMD genomewide association study (GWAS). In this comprehensive investigation of the skeletal genetic background in postmenopausal women, we identified functional bone ncRNAs associated to fracture and BMD, representing distinct subsets in WB and NWB skeletal sites. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Calmodulin-dependent kinase 1beta is expressed in the epiphyseal growth plate and regulates proliferation of mouse calvarial osteoblasts in vitro.

The Ca(2+)/Calmodulin-dependent protein kinase (CaMK) family is activated in response to elevation of intracellular Ca(2+), and includes CaMK1 (as well as CaMK2 and CaMK4), which exists as different isoforms (alpha, beta, gamma and delta). CaMK1 is present in several cell types and may be involved in various cellular processes, but its role in bone is unknown. In situ hybridization was used to determine the spatial and temporal expression of CaMK1beta during endochondral bone development in mouse embryos and newborn pups. The cellular and subcellular distribution of CaMK1 was assessed by quantitative immunogold electron microscopy (EM). The role of CaMK1beta in mouse calvarial osteoblasts was investigated by using small interfering RNA (siRNA) to silence its expression, while in parallel monitoring cell proliferation and levels of skeletogenic transcripts. cRNA in situ hybridization and EM studies show that CaMK1beta is mainly located in developing long bones and vertebrae (from ED14.5 until day 10 after birth), with highest expression in epiphyseal growth plate hypertrophic chondrocytes. By RT-PCR, we show that CaMK1beta2 (but not beta1) is expressed in mouse hind limbs (in vivo) and mouse calvarial osteoblasts (in vitro), and also in primary human articular chondrocyte cultures. Silencing of CaMK1beta in mouse calvarial osteoblasts by siRNA significantly decreases osteoblast proliferation and c-Fos gene expression (approx. 50%), without affecting skeletogenic markers for more differentiated osteoblasts (i.e. Cbfa1/Runx2, Osterix (Osx), Osteocalcin (Oc), Alkaline phosphatase (Alp) and Osteopontin (Opn)). These results identify CaMK1beta as a novel regulator of osteoblast proliferation, via mechanisms that may at least in part involve c-Fos, thus implicating CaMK1beta in the regulation of bone and cartilage development.

Distinct DNA methylation profiles in bone and blood of osteoporotic and healthy postmenopausal women.

DNA methylation affects expression of associated genes and may contribute to the missing genetic effects from genome-wide association studies of osteoporosis. To improve insight into the mechanisms of postmenopausal osteoporosis, we combined transcript profiling with DNA methylation analyses in bone. RNA and DNA were isolated from 84 bone biopsies of postmenopausal donors varying markedly in bone mineral density (BMD). In all, 2529 CpGs in the top 100 genes most significantly associated with BMD were analyzed. The methylation levels at 63 CpGs differed significantly between healthy and osteoporotic women at 10% false discovery rate (FDR). Five of these CpGs at 5% FDR could explain 14% of BMD variation. To test whether blood DNA methylation reflect the situation in bone (as shown for other tissues), an independent cohort was selected and BMD association was demonstrated in blood for 13 of the 63 CpGs. Four transcripts representing inhibitors of bone metabolism-MEPE, SOST, WIF1, and DKK1-showed correlation to a high number of methylated CpGs, at 5% FDR. Our results link DNA methylation to the genetic influence modifying the skeleton, and the data suggest a complex interaction between CpG methylation and gene regulation. This is the first study in the hitherto largest number of postmenopausal women to demonstrate a strong association among bone CpG methylation, transcript levels, and BMD/fracture. This new insight may have implications for evaluation of osteoporosis stage and susceptibility.

Methylation of bone SOST, its mRNA, and serum sclerostin levels correlate strongly with fracture risk in postmenopausal women.

Inhibition of sclerostin, a glycoprotein secreted by osteocytes, offers a new therapeutic paradigm for treatment of osteoporosis (OP) through its critical role as Wnt/catenin signaling regulator. This study describes the epigenetic regulation of SOST expression in bone biopsies of postmenopausal women. We correlated serum sclerostin to bone mineral density (BMD), fractures, and bone remodeling parameters, and related these findings to epigenetic and genetic disease mechanisms. Serum sclerostin and bone remodeling biomarkers were measured in two postmenopausal groups: healthy (BMD T-score > -1) and established OP (BMD T-score < -2.5, with at least one low-energy fracture). Bone specimens were used to analyze SOST mRNAs, single nucleotide polymorphisms (SNPs), and DNA methylation changes. The SOST gene promoter region showed increased CpG methylation in OP patients (n = 4) compared to age and body mass index (BMI) balanced controls (n = 4) (80.5% versus 63.2%, p = 0.0001) with replication in independent cohorts (n = 27 and n = 36, respectively). Serum sclerostin and bone SOST mRNA expression correlated positively with age-adjusted and BMI-adjusted total hip BMD (r = 0.47 and r = 0.43, respectively; both p < 0.0005), and inversely to serum bone turnover markers. Five SNPs, one of which replicates in an independent population-based genomewide association study (GWAS), showed association with serum sclerostin or SOST mRNA levels under an additive model (p = 0.0016 to 0.0079). Genetic and epigenetic changes in SOST influence its bone mRNA expression and serum sclerostin levels in postmenopausal women. The observations suggest that increased SOST promoter methylation seen in OP is a compensatory counteracting mechanism, which lowers serum sclerostin concentrations and reduces inhibition of Wnt signaling in an attempt to promote bone formation.

Molecular heterogeneity in human osteosarcoma demonstrated by enriched mRNAs isolated by directional tag PCR subtraction cloning.

Directional tag PCR subtractive hybridization was applied to construct a cDNA library generated from three different human osteosarcoma (OS) target cell lines (OHS, SaOS-2 and KPDXM) from which normal osteoblast (NO) sequences were subtracted. After two consecutive subtractive steps more than 98% of the common mRNAs species were depleted, leading to effective enrichment of the remaining target sequences. After differential screening of 960 clones, 81 candidates were further studied by Northern blot analysis and 73 represented separate mRNA species. Fifty-three of these showed enriched mRNA levels, of which 36 represented known and 17 not previously published cDNAs or EST sequences. The mRNAs showed a 1.4- to 504-fold enrichment compared to the mRNA levels in NO cells. The known mRNAs are: Ribosomal protein S11, KSP-37, Tethering factor SEC34, FXYD6, Alpha enolase, G-s-alpha, GPR85, DAF, RPL35A, GIF, TAPA-1, ANAPC11, DCI, hsp27, MRPS7 homolog, eIF p110 subunit, DPH2L, HMG-14, FB1 protein, chondroitin-6-sulphonase, calgizzarin, RNA polymerase II subunit, RPL13A, DHS, gp96, HHP2, acidic ribosomal phosphoprotein P2, ANT-2, ARF1, AFG3L2, SKD3, phosphoglucoisomerase, GST pi, CKI gamma 2, DNA polymerase delta small subunit and TRAP delta. Sections of human osteosarcoma biopsies and a xenograft were studied by in situ analysis. Seven cDNAs highly expressed in Northern blot analysis were tested. Their in situ expression differed between the xenograft and human sections as did that of collagen I. In the xenograft made from one of the target cell lines (OHS), a fair to strong representation of 3 cloned mRNAs was observed while collagen I mRNA was not detectable. We conclude that the molecular heterogeneity of these tumors is considerable. These results ought to have implications for future work to describe phenotypic subtypes with the aim of improving the diagnosis of human osteosarcomas.

Identification of transcriptional macromolecular associations in human bone using browser based in silico analysis in a giant correlation matrix.

Intracellular signaling is critically dependent on gene regulatory networks comprising physical molecular interactions. Presently, there is a lack of comprehensive databases for most human tissue types to verify such macromolecular interactions. We present a user friendly browser which helps to identify functional macromolecular interactions in human bone as significant correlations at the transcriptional level. The molecular skeletal phenotype has been characterized by transcriptome analysis of iliac crest bone biopsies from 84 postmenopausal women through quantifications of ~23,000 mRNA species. When the signal levels were inter-correlated, an array containing >260 million correlations was generated, thus recognizing the human bone interactome at the RNA level. The matrix correlation and p values were made easily accessible by a freely available online browser. We show that significant correlations within the giant matrix are reproduced in a replica set of 13 male vertebral biopsies. The identified correlations differ somewhat from transcriptional interactions identified in cell culture experiments and transgenic mice, thus demonstrating that care should be taken in extrapolating such results to the in vivo situation in human bone. The current giant matrix and web browser are a valuable tool for easy access to the human bone transcriptome and molecular interactions represented as significant correlations at the RNA-level. The browser and matrix should be a valuable hypothesis generating tool for identification of regulatory mechanisms and serve as a library of transcript relationships in human bone, a relatively inaccessible tissue.

Molecular disease map of bone characterizing the postmenopausal osteoporosis phenotype.

Genome-wide gene expressions in bone biopsies from patients with postmenopausal osteoporosis and healthy controls were profiled, to identify osteoporosis candidate genes. All osteoporotic patients (n = 27) in an unbiased cohort of Norwegian women presented with bone mineral density (BMD) T-scores of less than -2.5 SD and one or more confirmed low-energy fracture(s). A validation group (n = 18) had clinical and laboratory parameters intermediate to the control (n = 39) and osteoporosis groups. RNA from iliac crest bone biopsies were analyzed by Affymetrix microarrays and real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Differentially expressed genes in osteoporosis versus control groups were identified using the Bayesian ANOVA for microarrays (BAMarray) method, whereas the R-package Limma (Linear Models for Microarray Data) was used to determine whether these transcripts were explained by disease, age, body mass index (BMI), or combinations thereof. Laboratory tests showed normal ranges for the cohort. A total of 609 transcripts were differentially expressed in osteoporotic patients relative to controls; 256 transcripts were confirmed for disease when controlling for age or BMI. Most of the osteoporosis susceptibility genes (80%) also were confirmed to be regulated in the same direction in the validation group. Furthermore, 217 of 256 transcripts were correlated with BMD (adjusted for age and BMI) at various skeletal sites (|r| > 0.2, p < .05). Among the most distinctly expressed genes were Wnt antagonists DKK1 and SOST, the transcription factor SOX4, and the bone matrix proteins MMP13 and MEPE, all reduced in osteoporosis versus control groups. Our results identify potential osteoporosis susceptibility candidate genes adjusted for confounding factors (ie, age and BMI) with or without a significant correlation with BMD.

Periostin is a collagen associated bone matrix protein regulated by parathyroid hormone.

Periostin is a 90 kDa secreted protein, originally identified in murine osteoblast-like cells, with a distribution restricted to collagen-rich tissues and certain tumors. In this paper, we first analyzed the expression of periostin mRNA and protein in human fetal osteoblasts (hFOB) and human osteosarcoma (hOS) cell lines by RT real-time PCR and Western blot, respectively. The hFOB 1.19 and three hOS (MHM, KPDXM and Eggen) showed highly variable periostin mRNA levels and protein. Second, we showed that the expression of periostin mRNA was inversely related to the cells' abilities to differentiate and mineralize. Then, we investigated the regulation of periostin mRNA in hFOB after siRNA treatment and in mouse primary osteoblasts (mOB) treated with PTH. Knock-down of periostin mRNA, down-regulated PTHrP, but did not affect the expression of other important markers of differentiation such as RUNX2. In addition, periostin mRNA was transiently up-regulated in osteoblasts by PTH. Finally, the localization of periostin and its partially co-localization with collagen 1a1 mRNA and protein was studied in mouse embryos and postnatal pups using in situ hybridization and immunohistochemistry, respectively. In conclusion, the present study provides novel observations related to the expression, distribution and regulation of periostin in bone cells and extracellular matrix.

Eight genes are highly associated with BMD variation in postmenopausal Caucasian women.

Low bone mineral density (BMD) is an important risk factor for skeletal fractures which occur in about 40% of women >/=50 years in the western world. We describe the transcriptional changes in 84 trans-iliacal bone biopsies associated with BMD variations in postmenopausal females (50 to 86 years), aiming to identify genetic determinants of bone structure. The women were healthy or having a primary osteopenic or osteoporotic status with or without low energy fractures. The total cohort of 91 unrelated women representing a wide range of BMDs, were consecutively registered and submitted to global gene Affymetrix microarray expression analysis or histomorphometry. Among almost 23,000 expressed transcripts, a set represented by ACSL3 (acyl-CoA synthetase long-chain family member 3), NIPSNAP3B (nipsnap homolog 3B), DLEU2 (Deleted in lymphocytic leukemia, 2), C1ORF61 (Chromosome 1 open reading frame 61), DKK1 (Dickkopf homolog 1), SOST (Sclerostin), ABCA8, (ATP-binding cassette, sub-family A, member 8), and uncharacterized (AFFX-M27830-M-at), was significantly correlated to total hip BMD (5% false discovery rate) explaining 62% of the BMD variation expressed as T-score, 53% when adjusting for the influence of age (Z-score) and 44% when further adjusting for body mass index (BMI). Only SOST was previously associated to BMD, and the majority of the genes have previously not been associated with a bone phenotype. In molecular network analyses, SOST shows a strong, positive correlation with DKK1, both being members of the Wnt signaling pathway. The results provide novel insight in the underlying biology of bone metabolism and osteoporosis which is the ultimate consequence of low BMD.

Skeletal site-related variation in human trabecular bone transcriptome and signaling.

BACKGROUND: The skeletal site-specific influence of multiple genes on bone morphology is recognised, but the question as to how these influences may be exerted at the molecular and cellular level has not been explored. METHODOLOGY: To address this question, we have compared global gene expression profiles of human trabecular bone from two different skeletal sites that experience vastly different degrees of mechanical loading, namely biopsies from iliac crest and lumbar spinal lamina. PRINCIPAL FINDINGS: In the lumbar spine, compared to the iliac crest, the majority of the differentially expressed genes showed significantly increased levels of expression; 3406 transcripts were up- whilst 838 were down-regulated. Interestingly, all gene transcripts that have been recently demonstrated to be markers of osteocyte, as well as osteoblast and osteoclast-related genes, were markedly up-regulated in the spine. The transcriptome data is consistent with osteocyte numbers being almost identical at the two anatomical sites, but suggesting a relatively low osteocyte functional activity in the iliac crest. Similarly, osteoblast and osteoclast expression data suggested similar numbers of the cells, but presented with higher activity in the spine than iliac crest. This analysis has also led to the identification of expression of a number of transcripts, previously known and novel, which to our knowledge have never earlier been associated with bone growth and remodelling. CONCLUSIONS AND SIGNIFICANCE: This study provides molecular evidence explaining anatomical and micro-architectural site-related changes in bone cell function, which is predominantly attributable to alteration in cell transcriptional activity. A number of novel signaling molecules in critical pathways, which have been hitherto not known to be expressed in bone cells of mature vertebrates, were identified.

Osteopenia, decreased bone formation and impaired osteoblast development in Sox4 heterozygous mice.

The transcription factor Sox4 is vital for fetal development, as Sox4(-/-) homozygotes die in utero. Sox4 mRNA is expressed in the early embryonic growth plate and is regulated by parathyroid hormone, but its function in bone modeling/remodeling is unknown. We report that Sox4(+/-) mice exhibit significantly lower bone mass (by dual-energy X-ray absorptiometry) from an early age, and fail to obtain the peak bone mass of wild-type (WT) animals. Microcomputed tomography (muCT), histomorphometry and biomechanical testing of Sox4(+/-) bones show reduced trabecular and cortical thickness, growth plate width, ultimate force and stiffness compared with WT. Bone formation rate (BFR) in 3-month-old Sox4(+/-) mice is 64% lower than in WT. Primary calvarial osteoblasts from Sox4(+/-) mice demonstrate markedly inhibited proliferation, differentiation and mineralization. In these cultures, osterix (Osx) and osteocalcin (OCN) mRNA expression was reduced, whereas Runx2 mRNA was unaffected. No functional defects were found in osteoclasts. Silencing of Sox4 by siRNA in WT osteoblasts replicated the defects observed in Sox4(+/-) cells. We demonstrate inhibited formation and altered microarchitecture of bone in Sox4(+/-) mice versus WT, without apparent defects in bone resorption. Our results implicate the transcription factor Sox4 in regulation of bone formation, by acting upstream of Osx and independent of Runx2.

Butyrate response factor 1 is regulated by parathyroid hormone and bone morphogenetic protein-2 in osteoblastic cells.

Parathyroid hormone (PTH) exerts potent and diverse effects in bone and cartilage through activation of type 1 PTH receptors (PTH1R) capable of coupling to protein kinase A (PKA) and PKC. We have used macroarrays to identify zinc finger protein butyrate response factor-1 (BRF1) as a novel PTH regulated gene in clonal and normal osteoblasts of human and rodent origin. We further demonstrate that in human osteoblast-like OHS cells, biologically active hPTH(1-84) and hPTH(1-34) stimulate BRF1 mRNA expression in a dose- and time-dependent manner, while the amino-terminally truncated hPTH(3-84) which does not activate PTH1R has no effect. Moreover, using specific stimulators or inhibitors of PKA and PKC activity, the PTH-elicited BRF1 mRNA expression is mediated through the PKA signaling pathway. In mouse calvarial osteoblasts, BRF1 mRNA levels are upregulated by PTH(1-84) and reduced in response to bone morphogenetic protein 2 (BMP-2). Hence, our data showing that BRF1 is expressed in osteoblastic cells and regulated by PTH and BMP-2, suggest an important role for BRF1 in osteoblasts within the molecular network of PTH-dependent bone remodeling.