Three major signaling pathways have been identified to govern bone regeneration with an intense intracellular crosstalk: a) Bone morphogenetic protein (BMP) signaling, b) WNT signaling and c) signaling through parathyroid hormone receptor (PTH1R) activation. Recent research has highlighted the relevance of inhibitors of the respective pathways for the regulation of bone mass and thereby suggested new targets for the treatment of bone loss [1]. BMP proteins belong to the TGFb superfamily and activation of BMP receptors leads to induction of transcription through either MAP kinase signaling or phosphorylation of SMAD1/5/8 proteins [10,11]. Signaling through BMP proteins is regulated by either extracellular antagonists such as Noggin and Gremlin [12,13] or by intracellular inhibitors, e.g. inhibitory SMAD proteins [14] or nuclear MAB21L2 (Mab-21-like 2), a recently discovered BMP4 inhibitor [15]. Depending on coreceptors WNT signaling can be divided into canonical and non-canonical pathways. Canonical signaling is induced by binding of WNT ligands to the receptors of the Frizzled (FZD) family and LRP5/6 coreceptors, which results in activation of WNT-specific gene transcription by stabilization and nuclear translocation of b-Catenin. Non-canonical WNT signaling is transduced through FZD and ROR2/RYK coreceptors, which leads to the activation of G-protein or Ca2+-dependent cascades [16]. In MSC canonical signaling through WNT2, WNT3 or WNT3a induces proliferation and keeps the cells in an undifferentiated state, whereas non-canonical signaling, e.g. by WNT5a, WNT5b or WNT11, supports osteogenesis [17,18,19]. The osteocyte-specific factor Sclerostin (SOST) was described as an inhibitor of canonical WNT signaling, whereas there is ongoing discussion about its putative inhibitory effect on BMP signaling [20,21].
Sclerostin leads to reduced bone formation [22] and loss of function mutations are responsible for the high bone mass syndromes Van Buchem disease and sclerosteosis [23]. A neutralizing antibody against Sclerostin is a new, upcoming therapeutic treatment for osteoporosis [1,24]. Intermittent treatment with parathyroid hormone (PTH) is another therapeutical approach for osteoporosis and activates the third major signaling pathway in bone regeneration. However, continuous activation of PTH receptor has negative effects on bone homeostasis because subsequently enhanced RANKL expression on maturing osteoblasts stimulates osteoclast formation and bone resorption [25,26]. Interestingly, the genetic loci of proteins involved in the signaling pathways mentioned above, e.g. LRP5, LRP4, Sclerostin, PTH, BMPs or BMP receptor BMPR1B, have already been linked to the polygenetic nature of primary osteoporosis by wholegenome association studies and meta-analyses [27,28,29,30]. Besides genetic predisposition, advanced age is another strong risk factor for developing osteoporosis with adult stem cells being the restrictive parameter for unlimited tissue regeneration. In vitro, cells exhibit limited dividing capacity and enter replicative senescence, a state of irreversible G1 phase arrest, after about 50 population doublings [31,32]. It is caused by multiple factors like telomere shortening, oxidative stress, deficiencies in DNA repair and epigenetic changes. Currently it is still controversial, whether clock-driven, organismic aging is caused by the loss of selfregeneration due to replicative senescence of stem cells or by extrinsic environmental factors [33]. The impact of presumptive deficiencies of hMSC in elderly, osteoporotic patients has not been studied intensely yet and to ourknowledge changes at the gene expression level have not been examined before. Therefore, we performed microarray analyses of hMSC of elderly donors with and without osteoporosis to detect disease-associated changes in gene expression. With osteoporosis being an age-related disease, we also investigated the impact of aging on hMSC in general by analyzing the transcriptome of in vivo-aged and in vitro-aged, senescent cells. We discovered that hMSC of patients suffering from severe osteoporosis display a disease-specific gene expression pattern that is distinct from the effects of organismic aging per se. Besides the induced expression of inhibitors of bone formation we detected promising new candidate genes for osteoporosis and even found evidence for reduced stem cell function.
Results Osteoporosis-induced changes in gene expression
In this study, we compared the transcriptome of hMSC from 5 patients (79?4 years old) suffering from primary osteoporosis (hMSC-OP) with hMSC of the age-matched control group (hMSC-old; donor age 79?9 years) (Table 1). Genome-wide gene expression patterns were examined by employing microarray hybridizations; the obtained data was compared by SAM method (GEO accession number GSE35958). Fold changes (FC) in gene expression were regarded as significant at a threshold of at least 2fold and a false discovery rate (FDR) of less than 10%. We detected 2477 gene products with higher and 1222 gene products with reduced expression in osteoporotic hMSC-OP in comparison to non-osteoporotic hMSC-old (Figure 1A, Table S1). Osteoporosis as a polygenetic disease has been studied intensively on gene level, resulting in the detection of gene loci and polymorphisms associated with low bone mineral density (BMD), osteoporosis and fracture risk. In contrast to these approaches, our data represents the effects of both genetic and epigenetic changes in hMSC during the development of osteoporosis. To see if our results coincide at least partly with the genes associated to BMD by specific single nucleotide polymorphisms (SNP) and copy number variations, we searched the NCBI data base for genome-wide association studies, meta-analyses and candidate gene association studies. The genes listed in these studies were compared to all gene products differentially expressed in the approach hMSC-OP versus hMSC-old. We identified enhanced expression of 39 genes in hMSC-OP and reduced expression of 16 genes that are already described as reliable or promising candidates for osteoporosis, including susceptibility genes like LRP5, SPP1 (Osteopontin), COL1A1 and SOST (Table 2).
