The PPARgamma-selective ligand BRL-49653 differentially regulates the fate choices of rat calvaria versus rat bone marrow stromal cell populations

Abstract Background Osteoblasts and adipocytes are derived from a common mesenchymal progenitor and an inverse relationship between expression of the two lineages is seen with certain experimental manipulations and in certain diseases, i.e., osteoporosis, but the cellular pathway(s) and developmental stages underlying the inverse relationship is still under active investigation. To determine which precursor mesenchymal cell types can differentiate into adipocytes, we compared the effects of BRL-49653 (BRL), a selective ligand for peroxisome proliferators-activated receptor (PPAR)γ, a master transcription factor of adipogenesis, on osteo/adipogeneis in two different osteoblast culture models: the rat bone marrow (RBM) versus the fetal rat calvaria (RC) cell system. Results BRL increased the number of adipocytes and corresponding marker expression, such as lipoprotein lipase, fatty acid-binding protein (aP2), and adipsin, in both culture models, but affected osteoblastogenesis only in RBM cultures, where a reciprocal decrease in bone nodule formation and osteoblast markers, e.g., osteopontin, alkaline phosphatase (ALP), bone sialoprotein, and osteocalcin was seen, and not in RC cell cultures. Even though adipocytes were histologically undetectable in RC cultures not treated with BRL, RC cells expressed PPAR and CCAAT/enhancer binding protein (C/EBP) mRNAs throughout osteoblast development and their expression was increased by BRL. Some single cell-derived BRL-treated osteogenic RC colonies were stained not only with ALP/von Kossa but also with oil red O and co-expressed the mature adipocyte marker adipsin and the mature osteoblast marker OCN, as well as PPAR and C/EBP mRNAs. Conclusion The data show that there are clear differences in the capacity of BRL to alter the fate choices of precursor cells in stromal (RBM) versus calvarial (RC) cell populations and that recruitment of adipocytes can occur from multiple precursor cell pools (committed preadipocyte pool, multi-/bipotential osteo-adipoprogenitor pool and conversion of osteoprogenitor cells or osteoblasts into adipocytes (transdifferentiation or plasticity)). They also show that mechanisms beyond activation of PPARγ by its ligand are required for changing the fate of committed osteoprogenitor cells and/or osteoblasts into adipocytes

A Subset of Osteoblasts Expressing High Endogenous Levels of PPARγ Switches Fate to Adipocytes in the Rat Calvaria Cell Culture Model

Understanding fate choice and fate switching between the osteoblast lineage (ObL) and adipocyte lineage (AdL) is important to understand both the developmental inter-relationships between osteoblasts and adipocytes and the impact of changes in fate allocation between the two lineages in normal aging and certain diseases. The goal of this study was to determine when during lineage progression ObL cells are susceptible to an AdL fate switch by activation of endogenous peroxisome proliferator-activated receptor (PPAR)gamma.Multiple rat calvaria cells within the ObL developmental hierarchy were isolated by either fractionation on the basis of expression of alkaline phosphatase or retrospective identification of single cell-derived colonies, and treated with BRL-49653 (BRL), a synthetic ligand for PPARgamma. About 30% of the total single cell-derived colonies expressed adipogenic potential (defined cytochemically) when BRL was present. Profiling of ObL and AdL markers by qRT-PCR on amplified cRNA from over 160 colonies revealed that BRL-dependent adipogenic potential correlated with endogenous PPARgamma mRNA levels. Unexpectedly, a significant subset of relatively mature ObL cells exhibited osteo-adipogenic bipotentiality. Western blotting and immunocytochemistry confirmed that ObL cells co-expressed multiple mesenchymal lineage determinants (runt-related transcription factor 2 (Runx2), PPARgamma, Sox9 and MyoD which localized in the cytoplasm initially, and only Runx2 translocated to the nucleus during ObL progression. Notably, however, some cells exhibited both PPARgamma and Runx2 nuclear labeling with concomitant upregulation of expression of their target genes with BRL treatment.We conclude that not only immature but a subset of relatively mature ObL cells characterized by relatively high levels of endogenous PPARgamma expression can be switched to the AdL. The fact that some ObL cells maintain capacity for adipogenic fate selection even at relatively mature developmental stages implies an unexpected plasticity with important implications in normal and pathological bone development

IODP expedition 335 : deep sampling in ODP hole 1256D

Observations of the gabbroic layers of untectonized ocean crust are essential to test theoretical models of the accretion of new crust at mid-ocean ridges. Integrated Ocean Drilling Program (IODP) Expedition 335 ("Superfast Spreading Rate Crust 4") returned to Ocean Drilling Program (ODP) Hole 1256D with the intention of deepening this reference penetration of intact ocean crust a significant distance (~350 m) into cumulate gabbros. Three earlier cruises to Hole 1256D (ODP 206, IODP 309/312) have drilled through the sediments, lavas, and dikes and 100 m into a complex dike-gabbro transition zone. Operations on IODP Expedition 335 proved challenging throughout, with almost three weeks spent re-opening and securing unstable sections of the hole. When coring commenced, the comprehensive destruction of the coring bit required further remedial operations to remove junk and huge volumes of accumulated drill cuttings. Hole-cleaning operations using junk baskets were successful, and they recovered large irregular samples that document a hitherto unseen sequence of evolving geological conditions and the intimate coupling between temporally and spatially intercalated intrusive, hydrothermal, contact-metamorphic, partial melting, and retrogressive processes. Hole 1256D is now clean of junk, and it has been thoroughly cleared of the drill cuttings that hampered operations during this and previous expeditions. At the end of Expedition 335, we briefly resumed coring before undertaking cementing operations to secure problematic intervals. To ensure the greatest scientific return from the huge efforts to stabilize this primary ocean lithosphere reference site, it would be prudent to resume the deepening of Hole 1256D in the nearest possible future while it is open to full depth.7 page(s