title: Loss-of-Function of HtrA1 Abrogates All-Trans Retinoic Acid-Induced Osteogenic Differentiation of Mouse Adipose-Derived Stromal Cells Through Deficiencies in p70S6K Activation.
abstract: All-trans retinoic acid (ATRA) is a potent inducer of osteogenic differentiation in mouse adipose-derived stromal cells (mASCs), although the underlying mechanisms responsible for its mode of action have yet to be completely elucidated. High temperature requirement protease A1 (HtrA1) is a newly recognized modulator of human multipotent stromal cell (MSC) osteogenesis and as such, may play a role in regulating ATRA-dependent osteogenic differentiation of mASCs. In this study, we assessed the influence of small interfering RNA (siRNA)-induced repression of HtrA1 production on mASC osteogenesis and examined its effects on ATRA-mediated mammalian target of rapamycin (mTOR) signaling. Inhibition of HtrA1 production in osteogenic mASCs resulted in a significant reduction of alkaline phosphatase activity and mineralized matrix formation. Western blot analyses revealed the rapid activation of Akt (Ser473) and p70S6K (Thr389) in ATRA-treated mASCs, and that levels of phosphorylated p70S6K were noticeably reduced in HtrA1-deficient mASCs. Further studies using mTOR inhibitor rapamycin and siRNA specific for the p70S6K gene Rps6kb1 confirmed ATRA-mediated mASC osteogenesis as being dependent on p70S6K activation. Finally, transfection of cells with a constitutively active rapamycin-resistant p70S6K mutant could restore the mineralizing capacity of HtrA1-deficient mASCs. These findings therefore lend further support for HtrA1 as a positive mediator of MSC osteogenesis and provide new insights into the molecular mode of action of ATRA in regulating mASC lineage commitment.
authors.affiliation: 1 Bone and Stem Cell Research Group, CABMM, University of Zurich , Zurich, Switzerland .|2 Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich , Zurich, Switzerland .
authors.forename: Stephan
authors.initials: S
authors.lastname: Glanz
authors.affiliation: 1 Bone and Stem Cell Research Group, CABMM, University of Zurich , Zurich, Switzerland .
authors.forename: Ali
authors.initials: A
authors.lastname: Mirsaidi
authors.affiliation: 1 Bone and Stem Cell Research Group, CABMM, University of Zurich , Zurich, Switzerland .
authors.forename: Cristina
authors.initials: C
authors.lastname: López-Fagundo
authors.affiliation: 1 Bone and Stem Cell Research Group, CABMM, University of Zurich , Zurich, Switzerland .|2 Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich , Zurich, Switzerland .
authors.forename: Gladys
authors.initials: G
authors.lastname: Filliat
authors.affiliation: 1 Bone and Stem Cell Research Group, CABMM, University of Zurich , Zurich, Switzerland .
authors.forename: André N
authors.initials: AN
authors.lastname: Tiaden
authors.affiliation: 1 Bone and Stem Cell Research Group, CABMM, University of Zurich , Zurich, Switzerland .|2 Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich , Zurich, Switzerland .
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Richards
mesh_terms: D000273:Adipose Tissue / Q000166:cytology*; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D000074289:High-Temperature Requirement A Serine Peptidase 1; D051379:Mice; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D010012:Osteogenesis / Q000187:drug effects*; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000172:deficiency* / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D014212:Tretinoin; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D000074289:High-Temperature Requirement A Serine Peptidase 1; C492073:HtrA1 protein, mouse; D012697:Serine Endopeptidases; D020123:Sirolimus