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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 11β-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. 11β-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 11β-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 11β-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 11β-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.
Organic cation transporter 1 (OCT1, SLC22A1) is localized in the sinusoidal membrane of human hepatocytes and mediates hepatic uptake of weakly basic or cationic drugs and endogenous compounds. Common amino acid substitutions in OCT1 were associated with altered pharmacokinetics and efficacy of drugs like sumatriptan and fenoterol. Recently, the common splice variant rs35854239 has also been suggested to affect OCT1 function. rs35854239 represents an 8 bp duplication of the donor splice site at the exon 7-intron 7 junction. Here we quantified the extent to which this duplication affects OCT1 splicing and, as a consequence, the expression and the function of OCT1. We used pyrosequencing and deep RNA-sequencing to quantify the effect of rs35854239 on splicing after minigene expression of this variant in HepG2 and Huh7 cells and directly in human liver samples. Further, we analyzed the effects of rs35854239 on OCT1 mRNA expression in total, localization and activity of the resulting OCT1 protein, and on the pharmacokinetics of sumatriptan and fenoterol. The 8 bp duplication caused alternative splicing in 38% (deep RNA-sequencing) to 52% (pyrosequencing) of the minigene transcripts when analyzed in HepG2 and Huh7 cells. The alternatively spliced transcript encodes for a truncated protein that after transient transfection in HEK293 cells was not localized in the plasma membrane and was not able to transport the OCT1 model substrate ASP+. In human liver, however, the alternatively spliced OCT1 transcript was detectable only at very low levels (0.3% in heterozygous and 0.6% in homozygous carriers of the 8 bp duplication, deep RNA-sequencing). The 8 bp duplication was associated with a significant reduction of OCT1 expression in the human liver, but explained only 9% of the general variability in OCT1 expression and was not associated with significant changes in the pharmacokinetics of sumatriptan and fenoterol. Therefore, the rs35854239 variant only partially changes splicing, causing moderate changes in OCT1 expression and may be of only limited therapeutic relevance.