An osteocalcin-deficient mouse strain without endocrine abnormalities

Osteocalcin (OCN), the most abundant noncollagenous protein in the bone matrix, is reported to be a bone-derived endocrine hormone with wide-ranging effects on many aspects of physiology, including glucose metabolism and male fertility. Many of these observations were made using an OCN-deficient mouse allele (Osc– ) in which the 2 OCN-encoding genes in mice, Bglap and Bglap2, were deleted in ES cells by homologous recombination. Here we describe mice with a new Bglap and Bglap2 double-knockout (dko) allele (Bglap/2p.Pro25fs17Ter) that was generated by CRISPR/Cas9-mediated gene editing. Mice homozygous for this new allele do not express full-length Bglap or Bglap2 mRNA and have no immunodetectable OCN in their serum. FTIR imaging of cortical bone in these homozygous knockout animals finds alterations in the collagen maturity and carbonate to phosphate ratio in the cortical bone, compared with wild-type littermates. However, μCT and 3-point bending tests do not find differences from wild-type littermates with respect to bone mass and strength. In contrast to the previously reported OCN-deficient mice with the Osc− allele, serum glucose levels and male fertility in the OCN-deficient mice with the Bglap/ 2pPro25fs17Ter allele did not have significant differences from wild-type littermates. We cannot explain the absence of endocrine effects in mice with this new knockout allele. Possible explanations include the effects of each mutated allele on the transcription of neighboring genes, or differences in genetic background and environment. So that our findings can be confirmed and extended by other interested investigators, we are donating this new Bglap and Bglap2 double-knockout strain to the Jackson Laboratories for academic distribution

Kinome Profiling of NF1-Related MPNSTs in Response to Kinase Inhibition and Doxorubicin Reveals Therapeutic Vulnerabilities

Neurofibromatosis Type 1 (NF1)-related Malignant Peripheral Nerve Sheath Tumors (MPNST) are highly resistant sarcomas that account for significant mortality. The mechanisms of therapy resistance are not well-understood in MPNSTs, particularly with respect to kinase inhibition strategies. In this study, we aimed to quantify the impact of both the genomic context and targeted therapy on MPNST resistance using reverse phase phosphoproteome array (RPPA) analysis. We treated tumorgrafts from three genetically engineered mouse models using MET (capmatinib) and MEK (trametinib) inhibitors and doxorubicin, and assessed phosphosignaling at 4 h, 2 days, and 21 days. Baseline kinase signaling in our mouse models recapitulated an MET-addicted state (NF1-MET), P53 mutation (NF1-P53), and HGF overexpression (NF1). Following perturbation with the drug, we observed broad and redundant kinome adaptations that extended well beyond canonical RAS/ERK or PI3K/AKT/mTOR signaling. MET and MEK inhibition were both associated with an initial inflammatory response mediated by kinases in the JAK/STAT pathway and NFkB. Growth signaling predominated at the 2-day and 21-day time points as a result of broad RTK and intracellular kinase activation. Interestingly, AXL and NFkB were strongly activated at the 2-day and 21-day time points, and tightly correlated, regardless of the treatment type or genomic context. The degree of kinome adaptation observed in innately resistant tumors was significantly less than the surviving fractions of responsive tumors that exhibited a latency period before reinitiating growth. Lastly, doxorubicin resistance was associated with kinome adaptations that strongly favored growth and survival signaling. These observations confirm that MPNSTs are capable of profound signaling plasticity in the face of kinase inhibition or DNA damaging agent administration. It is possible that by targeting AXL or NFkB, therapy resistance can be mitigated

Phosphorylation of TXNIP by AKT Mediates Acute Influx of Glucose in Response to Insulin

Growth factors, such as insulin, can induce both acute and long-term glucose uptake into cells. Apart from the rapid, insulin-induced fusion of glucose transporter (GLUT)4 storage vesicles with the cell surface that occurs in muscle and adipose tissues, the mechanism behind acute induction has been unclear in other systems. Thioredoxin interacting protein (TXNIP) has been shown to be a negative regulator of cellular glucose uptake. TXNIP is transcriptionally induced by glucose and reduces glucose influx by promoting GLUT1 endocytosis. Here, we report that TXNIP is a direct substrate of protein kinase B (AKT) and is responsible for mediating AKT-dependent acute glucose influx after growth factor stimulation. Furthermore, TXNIP functions as an adaptor for the basal endocytosis of GLUT4 in vivo, its absence allows excess glucose uptake in muscle and adipose tissues, causing hypoglycemia during fasting. Altogether, TXNIP serves as a key node of signal regulation and response for modulating glucose influx through GLUT1 and GLUT4

Inhibiting WNT secretion reduces high bone mass caused by Sost loss-of-function or gain-of-function mutations in Lrp5.

Proper regulation of Wnt signaling is critical for normal bone development and homeostasis. Mutations in several Wnt signaling components, which increase the activity of the pathway in the skeleton, cause high bone mass in human subjects and mouse models. Increased bone mass is often accompanied by severe headaches from increased intracranial pressure, which can lead to fatality and loss of vision or hearing due to the entrapment of cranial nerves. In addition, progressive forehead bossing and mandibular overgrowth occur in almost all subjects. Treatments that would provide symptomatic relief in these subjects are limited. Porcupine-mediated palmitoylation is necessary for Wnt secretion and binding to the frizzled receptor. Chemical inhibition of porcupine is a highly selective method of Wnt signaling inhibition. We treated three different mouse models of high bone mass caused by aberrant Wnt signaling, including homozygosity for loss-of-function in Sost, which models sclerosteosis, and two strains of mice carrying different point mutations in Lrp5 (equivalent to human G171V and A214V), at 3 months of age with porcupine inhibitors for 5–6 weeks. Treatment significantly reduced both trabecular and cortical bone mass in all three models. This demonstrates that porcupine inhibition is potentially therapeutic for symptomatic relief in subjects who suffer from these disorders and further establishes that the continued production of Wnts is necessary for sustaining high bone mass in these models

Transcriptome and DNA methylome analyses reveal underlying mechanisms for the racial disparity in uterine fibroids

Uterine fibroids (leiomyomas) affect Black women disproportionately in terms of prevalence, incidence, and severity of symptoms. The causes of this racial disparity are essentially unknown. We hypothesized that myometria of Black women are more susceptible to developing fibroids and examined the transcriptomic and DNA methylation profiles of myometria and fibroids from Black and White women for comparison. Myometrial samples cluster by race in both their transcriptome and DNA methylation profiles, whereas fibroid samples only cluster by race in the latter. More differentially expressed genes (DEGs) were detected in the Black and White myometrial sample comparison than in the fibroid comparison. Leiomyoma gene set expression analysis identified four clusters of DEGs, including a cluster of 24 genes with higher expression in myometrial samples from Black women. One of the DEGs in this group, VWF, was significantly hypomethylated at two CpG probes that are near a putative enhancer site in myometrial samples from Black women and in all fibroids and that correlate with VWF expression levels. These results suggest that the molecular basis for the disparity in fibroid disease between Black and White women could be found in the myometria before fibroid development and not in the fibroids themselves

Activation of DAF-16/FOXO by reactive oxygen species contributes to longevity in long-lived mitochondrial mutants in <i>Caenorhabditis elegans</i>

<div><p>Mild deficits in mitochondrial function have been shown to increase lifespan in multiple species including worms, flies and mice. Here, we study three <i>C</i>. <i>elegans</i> mitochondrial mutants (<i>clk-1</i>, <i>isp-1</i> and <i>nuo-6</i>) to identify overlapping genetic pathways that contribute to their longevity. We find that genes regulated by the FOXO transcription factor DAF-16 are upregulated in all three strains, and that the transcriptional changes present in these worms overlap significantly with the long-lived insulin-IGF1 signaling pathway mutant <i>daf-2</i>. We show that DAF-16 and multiple DAF-16 interacting proteins (MATH-33, IMB-2, CST-1/2, BAR-1) are required for the full longevity of all three mitochondrial mutants. Our results suggest that the activation of DAF-16 in these mutants results from elevated levels of reactive oxygen species. Overall, this work reveals an overlapping genetic pathway required for longevity in three mitochondrial mutants, and, combined with previous work, demonstrates that DAF-16 is a downstream mediator of lifespan extension in multiple pathways of longevity.</p></div

DAF-16 is required for the longevity of <i>clk-1</i>, <i>isp-1</i> and <i>daf-2</i> worms.

<p>The long lifespan of <i>clk-1</i> (<b>A</b>), <i>isp-1</i> (<b>B</b>) and <i>daf-2</i> (<b>C</b>) mutants is reduced to wild-type by the <i>daf-16(mu86)</i> deletion mutation. <b>D.</b> The <i>daf-16(m26)</i> point mutation also decreases <i>isp-1</i> lifespan but to a lesser extent than the <i>mu86</i> deletion mutation. P-value indicates significance of difference between control and <i>daf-16</i> mutation for the experimental strain. Data and N for the lifespan experiments are included in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007268#pgen.1007268.s010" target="_blank">S2 Table</a></b>.</p

Overlap of upregulated genes between long-lived mitochondrial mutants and <i>daf-2</i> mutants.

<p><b>A.</b> Among the genes upregulated in the long-lived mitochondrial mutant strains <i>clk-1</i>, <i>isp-1</i> and <i>nuo-6</i>, many of these genes (46%, 50% and 57% respectively) are also upregulated in <i>daf-2</i> mutants. <b>B.</b> A heat map showing the top DAF-16 responsive genes that are upregulated in <i>daf-2</i> mutants (genes included are from the meta-analysis of DAF-16 target genes performed by Tepper <i>et al</i>., 2013 that were significantly upregulated in our <i>daf-2</i> RNAseq data). Many of these genes are also upregulated in the long-lived mitochondrial mutant strains. mRNA for all strains was isolated from six biological replicates per strain and sequenced individually.</p

Reactive oxygen species cause nuclear localization of DAF-16 and activation of DAF-16 target genes.

<p><b>A.</b> Increasing ROS levels through treatment with either 4 mM paraquat (PQ) or 300 μM juglone results in the nuclear localization of DAF-16. Young adult worms were treated with 300 μM juglone for 2 hours or 4 mM paraquat for 1 day. Age-matched untreated worms exhibited no nuclear localization of DAF-16:GFP (controls for 4 mM paraquat treated worms are shown). In wild-type worms, treatment with 4mM paraquat results in upregulation of DAF-16 target genes <i>dod-3</i> (<b>B</b>), <i>mtl-1</i> (<b>C</b>), <i>sodh-1</i> (<b>D</b>), and <i>ftn-1</i> (<b>E</b>). In contrast, this increase in gene expression resulting from elevated ROS is markedly reduced in the <i>daf-16(mu86)</i> deletion mutant (<b>B-E</b>)<b>. F.</b> As in the long-lived mitochondrial mutants and worms treated with 4 mM paraquat, multiple DAF-16 target genes are upregulated in <i>sod-2</i> deletion mutants. <b>G.</b> The upregulation of the DAF-16 target genes in <i>sod-2</i> worms is dependent on DAF-16 as knocking down <i>daf-16</i> mRNA using RNAi decreased the upregulation of DAF-16 target genes in <i>sod-2</i> worms. <b>H.</b> <i>daf-16</i> RNAi decreases the long lifespan of <i>sod-2</i> worms, indicating that DAF-16 is required for their longevity. P-value indicates significance of difference between EV (red) and <i>daf-16</i> RNAi (purple) for <i>sod-2</i> worms. <b>I.</b> Activation of the DAF-16 target gene <i>sod-3</i> in <i>isp-1</i> and <i>nuo-6</i> worms was reduced by treatment with antioxidants: 10 mM ascorbic acid, 25 μM butylated hydroxyanisole (BHA) or 10 mM sodium ascorbate. Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001. Data and N for the lifespan experiments are included in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007268#pgen.1007268.s010" target="_blank">S2 Table</a></b>.</p