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.

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.

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.

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.

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.

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.

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.