Osteoporosis is a chronic skeletal disease characterised by reduced bone mass and bone fragility. It remains a major public health problem due to the high prevalence of the disease, late diagnosis and severe consequences of low-energy fractures. Genetic factors contribute to the incidence of osteoporosis and are also detected by the genome-wide association study approach (GWAS). Several GWAS studies have identified the membrane palmitoylated protein 7 (MPP7) gene as a candidate gene associated with reduced bone density, but its role in bone biology is not yet known.
The aim of this master thesis was to evaluate the effect of MPP7 gene knockout on the efficiency of mineralisation or extracelular mineralised matrix formation in a cellular model of human osteosarcoma HOS cells. We wanted to determine how MPP7 gene knockout affects the dynamics of expression of some key osteogenic factors during the osteogenic differentiation process. In this study we used pre-prepared and lysed human osteosarcoma HOS cell lines (ATCC® CRL-1543␢) with MPP7 gene knockout (MPP7-KO) and wild type (WT) HOS cells with functional MPP7 gene (HOS WT).
Expression of selected osteogenic factors was measured by real time quantitative polymerase chain reaction (RT-qPCR). We determined the effect on the expression of genes related to osteoblasts differenctiation RUNX2 (Runt-related transcription factor 2) gene and PPARG (peroxisome proliferator activated receptor gamma) gene. We also measured the effect on the expression of genes related to basic osteoblasts function, ALP (alkaline phosphatase) gene, COL1a1 (collagen type I, alpha chain I) gene and OC (osteocalcin) gene, as well as the intracellular Wnt signalling pathway genes CTNNB1 (β-catenin), CDH2 (N-kadherin) and CCND1 (Cyclin D1). The degree of mineralisation in both cell lines was determined qualitatively (microscopic analysis) and quantitatively (extraction of the dye with acetic acid and measurement of absorbance at 405 nm) by in vitro histochemical staining of fixed plates with Alizarin Red S, that binds to calcium mineral inclusions. Cell morphology was also observed by microscopic analysis.
Our results show that MPP7 plays a key role in bone mineralisation. Results of histochemical staining with Alizarin Red S showed that in HOS WT cells mineral inclusions stain during differentiation, while in MPP7-KO cells we did not detect formation of calcium deposits either qualitatively by microscopic analysis or quantitatively by absorbance measurement. We also observed the formation of circles from morphologically distinct cells only in the HOS WT cell line. Consistent with these results, ALP expression was completely suppressed in MPP7-KO cells. MPP7 knockout also affects the expression of the osteogenic factor RUNX2, which is more progressive in MPP7-KO cells, as well as the adipogenic transcription factor PPARG, whose expression is significantly increased in the absence of the MPP7 gene. MPP7 also directly affects Wnt signalling pathway as less CDH2 is expressed in MPP7-KO cells, making more active β-catenin available, which was consistent with higher expression of CCND1 (downstream target gene of β-catenin) in those cells.
In our thesis we show that MPP7 is essential for bone matrix mineralisation and ALP expression, influences the expression of major transcription factors RUNX2 and PPARG and directly affects the activity of the canonical Wnt signalling pathway.
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