A bone-specific adipogenesis pathway in fat-free mice defines key origins and adaptations of bone marrow adipocytes with age and disease

Bone marrow adipocytes accumulate with age and in diverse disease states. However, their origins and adaptations in these conditions remain unclear, impairing our understanding of their context-specific endocrine functions and relationship with surrounding tissues. In this study, by analyzing bone and adipose tissues in the lipodystrophic \u27fat-free\u27 mouse, we define a novel, secondary adipogenesis pathway that relies on the recruitment of adiponectin-negative stromal progenitors. This pathway is unique to the bone marrow and is activated with age and in states of metabolic stress in the fat-free mouse model, resulting in the expansion of bone marrow adipocytes specialized for lipid storage with compromised lipid mobilization and cytokine expression within regions traditionally devoted to hematopoiesis. This finding further distinguishes bone marrow from peripheral adipocytes and contributes to our understanding of bone marrow adipocyte origins, adaptations, and relationships with surrounding tissues with age and disease

Marrow adipose tissue expansion coincides with insulin resistance in MAGP1-deficient mice

Marrow adipose tissue (MAT) is an endocrine organ with the potential to influence skeletal remodeling and hematopoiesis. Pathologic MAT expansion has been studied in the context of severe metabolic challenge, including caloric restriction, high fat diet feeding, and leptin deficiency. However, the rapid change in peripheral fat and glucose metabolism associated with these models impedes our ability to examine which metabolic parameters precede or coincide with MAT expansion. Microfibril-associated glycoprotein-1 (MAGP1) is a matricellular protein that influences cellular processes by tethering signaling molecules to extracellular matrix structures. MAGP1-deficient (Mfap2(−/−)) mice display a progressive excess adiposity phenotype, which precedes insulin resistance and occurs without changes in caloric intake or ambulation. Mfap2(−/−) mice were, therefore, used as a model to associate parameters of metabolic disease, bone remodeling, and hematopoiesis with MAT expansion. Marrow adiposity was normal in Mfap2(−/−) mice until 6 months of age; however, by 10 months, marrow fat volume had increased fivefold relative to wild-type control at the same age. Increased gonadal fat pad mass and hyperglycemia were detectable in Mfap2(−/−) mice by 2 months, but peaked by 6 months. The development of insulin resistance coincided with MAT expansion. Longitudinal characterization of bone mass demonstrated a disconnection in MAT volume and bone volume. Specifically, Mfap2(−/−) mice had reduced trabecular bone volume by 2 months, but this phenotype did not progress with age or MAT expansion. Interestingly, MAT expansion in the 10-month-old Mfap2(−/−) mice was associated with modest alterations in basal hematopoiesis, including a shift from granulopoiesis to B lymphopoiesis. Together, these findings indicate MAT expansion is coincident with insulin resistance, but not excess peripheral adiposity or hyperglycemia in Mfap2(−/−) mice; and substantial MAT accumulation does not necessitate a proportional decrease in either bone mass or bone marrow cellularity

Sarm1 knockout prevents type 1 diabetic bone disease in females independent of neuropathy

Patients with diabetes have a high risk of developing skeletal diseases accompanied by diabetic peripheral neuropathy (DPN). In this study, we isolated the role of DPN in skeletal disease with global and conditional knockout models of sterile-α and TIR-motif-containing protein-1 (Sarm1). SARM1, an NADase highly expressed in the nervous system, regulates axon degeneration upon a range of insults, including DPN. Global knockout of Sarm1 prevented DPN, but not skeletal disease, in male mice with type 1 diabetes (T1D). Female wild-type mice also developed diabetic bone disease but without DPN. Unexpectedly, global Sarm1 knockout completely protected female mice from T1D-associated bone suppression and skeletal fragility despite comparable muscle atrophy and hyperglycemia. Global Sarm1 knockout rescued bone health through sustained osteoblast function with abrogation of local oxidative stress responses. This was independent of the neural actions of SARM1, as beneficial effects on bone were lost with neural conditional Sarm1 knockout. This study demonstrates that the onset of skeletal disease occurs rapidly in both male and female mice with T1D completely independently of DPN. In addition, this reveals that clinical SARM1 inhibitors, currently being developed for treatment of neuropathy, may also have benefits for diabetic bone through actions outside of the nervous system

The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling

The adenosine diphosphate (ADP) receptor P2RY12 (purinergic receptor P2Y, G protein coupled, 12) plays a critical role in platelet aggregation, and P2RY12 inhibitors are used clinically to prevent cardiac and cerebral thrombotic events. Extracellular ADP has also been shown to increase osteoclast (OC) activity, but the role of P2RY12 in OC biology is unknown. Here, we examined the role of mouse P2RY12 in OC function. Mice lacking P2ry12 had decreased OC activity and were partially protected from age-associated bone loss. P2ry12(–/–) OCs exhibited intact differentiation markers, but diminished resorptive function. Extracellular ADP enhanced OC adhesion and resorptive activity of WT, but not P2ry12(–/–), OCs. In platelets, ADP stimulation of P2RY12 resulted in GTPase Ras-related protein (RAP1) activation and subsequent α(IIb)β(3) integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin β(3) gene knockout (Itgb3(–/–)) OCs, but its effects were substantially blunted in P2ry12(–/–) OCs. In vivo, P2ry12(–/–) mice were partially protected from pathologic bone loss associated with serum transfer arthritis, tumor growth in bone, and ovariectomy-induced osteoporosis: all conditions associated with increased extracellular ADP. Finally, mice treated with the clinical inhibitor of P2RY12, clopidogrel, were protected from pathologic osteolysis. These results demonstrate that P2RY12 is the primary ADP receptor in OCs and suggest that P2RY12 inhibition is a potential therapeutic target for pathologic bone loss

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Title Page Title: Genistein induces phenotypic reversion of endoglin deficiency in human prostate cancer cells Running Title Page Running Title: Endoglin deficiency phenotype reversed by genistein Correspondence to

Abstract Genistein has been shown to inhibit human prostate cancer (PCa) cell motility. Endoglin has been identified as an important suppressor of PCa cell motility, and its expression is lost during PCa progression. It is therefore important to determine whether endoglin loss affects genistein&apos;s efficacy, and if so, by what mechanism. In the current study, genistein was shown to induce reversion of endoglin deficient cells to a low motility, endoglin replete, phenotype. As endoglin suppresses PCa cell motility in an ALK2 and Smad1 dependent manner, we sought to determine whether genistein was activating the ALK2-Smad1 pathway. While treatment with genistein, or over expression of Smad1 or ALK2, all increased Smad1 responsive promoter activity and decreased cell motility, genistein&apos;s efficacy was abrogated by either Smad1 or ALK2 knock down. Further, transfection of cells with a kinase dead mutant of ALK2 abrogated genistein&apos;s efficacy. Together, these findings demonstrate that genistein therapeutically induces reversion to a low motility phenotype in aggressive endoglin deplete PCa cells. It does so by activating ALK2-Smad1 endoglin-associated signaling. These findings support the notion that individuals with low endoglin expressing PCa will benefit from genistein treatment. MOL #38935 4 Prostate cancer (PCa) is a leading cause of cancer associated death in the United States and world wide We have previously demonstrated that genistein (4&apos;,5,7-trihydroxyflavone) inhibits PCa cell invasion Dysregulated cell motility is a basic characteristic of cancer, including PCa, and is seen during PCa progression. Molecular changes which relate to the regulation of cell motility underlie this abnormal cellular phenotype. To be effective, anti-cancer therapeutics must retain efficacy in the face of molecular aberrations associated with cancer progression. Alternatively, their use must be tailored to specific molecular profiles. In either situation, optimal therapeutic implementation requires an MOL #38935 5 understanding of the relationship between therapeutic intervention, and the underlying molecular profile. A series of prior studies by us have identified endoglin as a key regulator of PCa cell motility, and have shown that its expression is lost during PCa progression Endoglin is a 180 kDa homodimeric type I transmembrane auxiliary receptor in the TGFβ superfamily A consideration of a series of studies by us and others supports the notion that genistein may exert effects upon the endoglin signaling pathway. Both endoglin and genistein act to suppress PCa cell invasion. Further, we have recently demonstrated that endoglin suppresses PCa cell motility by activating Smad1, in an ALK2 dependent manner Smad1 promoter luciferase reporter assays. Cells were co-transfected with BRE2-Luc and β -gal, and luciferase and β -gal activity were measured as described Quantitative reverse transcription/polymerase chain reaction (qRT/PCR). RNA isolation and real time qRT/PCR were performed as described Given the small magnitude of the increase, and the lack of statistical significance, this increase was felt not to be responsible for genistein&apos;s effects. This notion is supported by other findings. First, genistein retained efficacy in the face of endoglin knock down. Second, though endoglin expression did not increase with increases in genistein concentration, we have previously shown enhanced anti-invasion efficacy by genistein across this concentration range As endoglin has been shown to suppress PCa cell invasion by activating Smad1, we hypothesized that genistein was activating Smad1 promoter activity was not altered by endoglin knock down. Genistein cooperates with Smad1 to inhibit ALK2 is necessary for genistein-mediated inhibition of cell invasion. Type I (RI) TGFβ superfamily receptors have kinase domains which function as activators of Smad proteins Further, these findings suggest that individuals with endoglin deficient PCa may in fact experience a greater therapeutic benefit from therapy, than those with normal endoglin expression. We also show for the first time that genistein compensates for endoglin deficiency by activating endoglin-associated signaling pathways. In particular, endoglin activated Smad1 transcriptional activity. This in turn was shown to require ALK2, and in particular, a kinase competent ALK2. We have recently shown that endoglin inhibits PCa cell motility through a mechanism involving the type I TGFβ superfamily receptor ALK2 and Smad1 18 treatment conditions, genistein decreases PCa cell invasion but not cell viability Finally, in the current study, cell viability was closely monitored, and was not adversely altered under experimental conditions, compared to relevant controls. We thus propose the schema depicted in MOL #38935 19 The current study identifies ALK2, and in particular, kinase competent ALK2 as necessary for genistein-mediated reversion to an endoglin replete phenotype. However, additional studies will be required to further elucidate the underlying mechanism. One possibility is that genistein may alter the molecular make up of heteromeric cell surface receptor complexes. Canonical signaling through TGFβ superfamily receptors requires the formation of a multi-protein cell surface complex which contains two or more RI subtypes, two or more RII subtypes, and with and without one or more endoglin subunits In summary, genistein was shown to induce reversion of low endoglin PCa cells to a low motility, high endoglin, phenotype. This was due to genistein-mediated activation of Smad1, which in turn was dependent upon kinase competent ALK2. As endoglin is lost during PCa progression, and contributes to its metastatic phenotype, the current study supports the notion that individuals with low endoglin expressing PCa may derive relatively high therapeutic benefit from genistein. These findings may help interpret ongoing phase II molecular efficacy studies of genistein in prostate and other cancers. MOL #38935 2