ABSTRACT
Osteoblasts and adipocytes are derived from a common precursor in bone marrow, the mesenchymal stem cell (MSC). Factors driving human MSCs (hMSCs) to differentiate down the two lineages play important roles in determining bone density because it has been shown that bone volume loss associated with osteoporosis and aging is accompanied by reduced osteoblastic bone formation and increased marrow adipose tissue. The genes upregulated in hMSCs during osteogenic differentiation were screened using cDNA microarrays and were semi-quantitated by real-time RT-PCR. One of the genes identified was sortilin, which was upregulated one day after osteogenic induction and remained upregulated for a week. The overexpression of sortilin in hMSCs using an adenovirus vector resulted in the acceleration of mineralization during osteogenic differentiation without affecting alkaline phosphatase activity. Lipoprotein lipase (LPL), produced by adipocytes, is bound by sortilin, which may mediate its endocytosis. By adding LPL to osteogenic induction medium, osteoblastic mineralization was inhibited in a dose-dependent manner. Interestingly, sortilin overexpression abolished the LPL-mediated suppression of osteogenic differentiation. hMSCs exist in marrow where LPL-producing adipose cells are abundant and where osteogenesis is negatively regulated by LPL. Sortilin has a counter effect of promoting osteogenesis by acting as a scavenger of LPL.
Subject(s)
Calcification, Physiologic/physiology , Extracellular Matrix/metabolism , Membrane Glycoproteins/metabolism , Mesoderm/metabolism , Nerve Tissue Proteins/metabolism , Osteoblasts/metabolism , Stem Cells/metabolism , Adaptor Proteins, Vesicular Transport , Alkaline Phosphatase/biosynthesis , Bone Marrow Cells/cytology , Calcification, Physiologic/drug effects , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cells, Cultured , Dose-Response Relationship, Drug , Extracellular Matrix/drug effects , Gene Expression Profiling , Humans , Lipoprotein Lipase/pharmacology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/pharmacology , Mesoderm/cytology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/pharmacology , Oligonucleotide Array Sequence Analysis , Osteoblasts/cytology , Osteoblasts/drug effects , Stem Cells/cytology , Stem Cells/drug effects , Transfection , Up-RegulationABSTRACT
Ossification of the posterior longitudinal ligament of the spine (OPLL) is the predominant myelopathy among Japanese, and is usually diagnosed by ectopic bone formation in the paravertebral ligament in Japanese and other Asians. To detect genetic determinants associated with OPLL, we performed an extensive nonparametric linkage study with 126 affected sib-pairs using markers for various candidate genes by distinct analyses, SIBPAL and GENEHUNTER. Eighty-eight candidate genes were selected by comparing the genes identified by complementary DNA (cDNA) microarray analysis of systematic gene expression profiles during osteoblastic differentiation of human mesenchymal stem cells with the genes known to be involved in bone metabolism. Of the 24 genes regulated during osteoblastic differentiation, only one, the alpha B crystalline gene, showed evidence of linkage (p = 0.016, nonparametric linkage [NPL] score = 1.83). Of 64 genes known to be associated with bone metabolism, 7 showed weak evidence of linkage by SIBPAL analysis (p < 0.05): cadherin 13 (CDH13), bone morphogenetic protein 4 (BMP4), proteoglycan 1 (PRG1), transforming growth factor beta 3 (TGFb3), osteopontin (OPN), parathyroid hormone receptor 1 (PTHR1), and insulin-like growth factor 1 (IGF1). Among these genes, BMP4 (NPL = 2.23), CDH13 (NPL = 2.00), TGFb3 (NPL = 1.30), OPN (NPL = 1.15), and PTHR1 (NPL = 1.00) showed evidence of linkage by GENEHUNTER. Only BMP4 reached criteria of suggestive evidence of linkage. Because this gene is a well-known factor in osteogenetic function, BMP4 should be screened in further study for the polymorphism responsible.
Subject(s)
Ossification of Posterior Longitudinal Ligament/genetics , Animals , Artificial Intelligence , Base Sequence , Bone Morphogenetic Protein 4 , Bone Morphogenetic Proteins/genetics , Case-Control Studies , Crystallins/genetics , Disease Models, Animal , Female , Gene Expression Profiling , Genetic Linkage , Genetic Testing , Humans , Male , Mice , Microsatellite Repeats , Middle Aged , Oligonucleotide Array Sequence Analysis , RatsABSTRACT
The extent and distribution of linkage disequilibrium ( LD) in humans is a current topic especially for gene mapping of complex diseases. Akaike's information criterion ( AIC) was applied to estimate LD and compared with other standard LD measures, D' and r(2). By comparison of an independent model (IM; linkage equilibrium) and a dependent model (DM; linkage disequilibrium), the parsimonious model is the one with the smaller AIC score. Therefore, the extent of LD by AIC is expressed as AIC( IM) -- AIC( DM)( AIC( LD)). A total of 39 single-nucleotide polymorphisms on a 1.6-Mb region of chromosome 21 q22 were identified, and genotyped in 192 Japanese individuals. All possible pairs were analyzed to estimate LD and the analyses were compared. AIC( LD) became highly positive as the D' value increased and was negative at D' values of around 0.2. Because a negative value of AIC( LD) implies linkage equilibrium, D' values below 0.2 should be regarded as linkage equilibrium. The LD estimate by AIC yielded results similar to those obtained by r(2), indicating that AIC( LD) would be useful for fine gene mapping.
