Genetic Variants in WNT16 and PKD2L1 Locus Affect Heel Ultrasound Bone Stiffness: Analyses from the General Population and Patients Evaluated for Osteoporosis.

Osteoporosis, a complex chronic disease with increasing prevalence, is characterised by reduced bone mineral density (BMD) and increased fracture risk. The high heritability of BMD suggests substantial impact of the individual genetic disposition on bone phenotypes and the development of osteoporosis. In the past years, genome-wide association studies (GWAS) identified hundreds of genetic variants associated with BMD or osteoporosis. Here, we analysed 1103 single nucleotide polymorphisms (SNPs), previously identified as associated with estimated BMD (eBMD) in the UK Biobank. We assessed whether these SNPs are related to heel stiffness index obtained by quantitative ultrasound in 5665 adult participants of the Study of Health in Pomerania (SHIP). We confirmed 45 significant associations after correction for multiple testing. Next, we analysed six selected SNPs in 631 patients evaluated for osteoporosis [rs2707518 (CPED1/WNT16), rs3779381 (WNT16), rs115242848 (LOC101927709/EN1), rs10239787 (JAZF1), rs603424 (PKD2L1) and rs6968704 (JAZF1)]. Differences in minor allele frequencies (MAF) of rs2707518 and rs3779381 between SHIP participants (higher MAF) and patients evaluated for osteoporosis (lower MAF) indicated a protective effect of the minor allele on bone integrity. In contrast, differences in MAF of rs603424 indicated a harmful effect. Co-localisation analyses indicated that the rs603424 effect may be mediated via stearoyl-CoA desaturase (SCD) expression, an enzyme highly expressed in adipose tissue with a crucial role in lipogenesis. Taken together, our results support the role of the WNT16 pathway in the regulation of bone properties and indicate a novel causal role of SCD expression in adipose tissue on bone integrity.

Effects of HSD11B1 knockout and overexpression on local cortisol production and differentiation of mesenchymal stem cells.

Exogenous glucocorticoids increase the risk for osteoporosis, but the role of endogenous glucocorticoids remains elusive. Here, we describe the generation and validation of a loss- and a gain-of-function model of the cortisol producing enzyme 11ß-HSD1 (HSD11B1) to modulate the endogenous glucocorticoid conversion in SCP-1 cells - a model for human mesenchymal stem cells capable of adipogenic and osteogenic differentiation. CRISPR-Cas9 was successfully used to generate a cell line carrying a single base duplication and a 5 bp deletion in exon 5, leading to missense amino acid sequences after codon 146. These inactivating genomic alterations were validated by deep sequencing and by cloning with subsequent capillary sequencing. 11ß-HSD1 protein levels were reduced by 70% in the knockout cells and cortisol production was not detectable. Targeted chromosomal integration was used to stably overexpress HSD11B1. Compared to wildtype cells, HSD11B1 overexpression resulted in a 7.9-fold increase in HSD11B1 mRNA expression, a 5-fold increase in 11ß-HSD1 protein expression and 3.3-fold increase in extracellular cortisol levels under adipogenic differentiation. The generated cells were used to address the effects of 11ß-HSD1 expression on adipogenic and osteogenic differentiation. Compared to the wildtype, HSD11B1 overexpression led to a 3.7-fold increase in mRNA expression of lipoprotein lipase (LPL) and 2.5-fold increase in lipid production under adipogenic differentiation. Under osteogenic differentiation, HSD11B1 knockout led to enhanced alkaline phosphatase (ALP) activity and mRNA expression, and HSD11B1 overexpression resulted in a 4.6-fold and 11.7-fold increase in mRNA expression of Dickkopf-related protein 1 (DKK1) and LPL, respectively. Here we describe a HSD11B1 loss- and gain-of-function model in SCP-1 cells at genetic, molecular and functional levels. We used these models to study the effects of endogenous cortisol production on mesenchymal stem cell differentiation and demonstrate an 11ß-HSD1 dependent switch from osteogenic to adipogenic differentiation. These results might help to better understand the role of endogenous cortisol production in osteoporosis on a molecular and cellular level.

The rise in expression and activity of 11ß-HSD1 in human mesenchymal progenitor cells induces adipogenesis through increased local cortisol synthesis.

11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) plays an important role in pre-receptor glucocorticoid metabolism. This enzyme is expressed in bone, increases with age, and catalyzes the conversion of the inactive glucocorticoid cortisone into the active glucocorticoid cortisol and vice versa. Here we hypothesized that the physiological activity of 11ß-HSD1 to produce cortisol in human mesenchymal progenitor cells (hMSC) is principally sufficient to shift the differentiation potential in the direction of adipogenic. We thus investigated differentiating hMSCs and the mesenchymal stem cell line SCP-1 cultured under osteogenic conditions and stimulated with supra-physiological cortisone levels. The release of active cortisol into the medium was monitored and the influence on cell differentiation analyzed. We revealed an increase in 11ß-HSD1 expression followed by increased reductive activity of the enzyme, thereby inducing a more adipogenic phenotype of the cell models via cortisol with negative effects on osteogenesis. Through inhibition experiments with the specific inhibitor 10 j, we proved the enzyme specificity for cortisol synthesis and adipogenic differentiation. Increased expression of 11ß-HSD1 followed by higher cortisol levels might thus explain bone marrow adiposity followed by reduced bone quality and stability in old age or in situations of supra-physiological glucocorticoid exposure.