SirT1 regulates bone mass in vivo through regulation of osteoblast and osteoclast differentiation

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2009.Includes bibliographical references.As mammals age, osteoblast numbers decrease while osteoclast numbers increase, resulting in age-related or post-menopausal osteoporosis. SirT1, the mammalian orthologue of yeast Sir2, has been shown to be a negative regulator of PPAR[gamma] and NF[kappa]B, two transcription factors important for the differentiation of osteoblasts and osteoclasts, respectively. Here, we show that SirT1 is an important regulator of bone mass in vivo: SirT1 whole-body knockout mice display significant bone deficiencies that are associated with decreased osteoblast and increased osteoclast numbers. Further, osteoblast (ObKO) and osteoclast (OcKO) specific SirT1 knockout mice also show an osteoporotic phenotype that is associated with decreased osteoblasts in the ObKOs and increased osteoclasts in the OcKOs. In osteoblasts, we find that instead of targeting PPAR[gamma], SirT1 interacts with and increases the transactivation potential of Runx2. In osteoclasts, SirT1 inhibits differentiation largely through repression of the p65/RelA subunit of NF[kappa]B. Finally, we show that calorie restriction (CR) results in increased bone mass that is associated with higher osteoblast and lower osteoclast numbers - the inverse phenotype of SirT1 knockout mice. We find CR induces expression and activity of SirT1 in bone, and that SirT1 knockout mice fail to show any increases in bone mass in response to CR. Our results therefore provide the first evidence that SirT1 is an important regulator of bone mass in vivo, possibly providing a link between bone remodeling, metabolism and osteoporosis.by Kayvan Zainabadi.Ph.D

SIRT1 is a positive regulator of in vivo bone mass and a therapeutic target for osteoporosis

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Overexpression or pharmacological activation of SIRT1 has been shown to extend the lifespan of mice and protect against aging-related diseases. Here we show that pharmacological activation of SIRT1 protects in two models of osteoporosis. Ovariectomized female mice and aged male mice, models for post-menopausal and aging-related osteoporosis, respectively, show significant improvements in bone mass upon treatment with SIRT1 agonist, SRT1720. Further, we find that calorie restriction (CR) results in a two-fold upregulation of sirt1 mRNA expression in bone tissue that is associated with increased bone mass in CR mice. Reciprocally, SIRT1 whole-body knockout (KO) mice, as well as osteoblast and osteoclast specific KOs, show a low bone mass phenotype; though double knockout mice (containing SIRT1 deleted in both osteoblasts and osteoclasts) do not show a more severe phenotype. Altogether, these findings provide strong evidence that SIRT1 is a positive regulator of bone mass and a promising target for the development of novel therapeutics for osteoporosis

Myeloid Cell Sirtuin-1 Expression Does Not Alter Host Immune Responses to Gram-Negative Endotoxemia or Gram-Positive Bacterial Infection

The role of sirtuin-1 (SIRT1) in innate immunity, and in particular the influence of SIRT1 on antimicrobial defense against infection, has yet to be reported but is important to define since SIRT1 inhibitors are being investigated as therapeutic agents in the treatment of cancer, Huntington’s disease, and autoimmune diseases. Given the therapeutic potential of SIRT1 suppression, we sought to characterize the role of SIRT1 in host defense. Utilizing both pharmacologic methods and a genetic knockout, we demonstrate that SIRT1 expression has little influence on macrophage and neutrophil antimicrobial functions. Myeloid SIRT1 expression does not change mortality in gram-negative toxin-induced shock or gram-positive bacteremia, suggesting that therapeutic suppression of SIRT1 may be done safely without suppression of myeloid cell-specific immune responses to severe bacterial infections.American Asthma Foundation (Stephen Silberstein Senior Fellowship Awards

One in Four Individuals of African-American Ancestry Harbors a 5.5kb Deletion at chromosome 11q13.1

Cloning and sequencing of 5.5kb deletion at chromosome 11q13.1 from the HeLa cells, tumorigenic hybrids and two fibroblast cell lines has revealed homologous recombination between AluSx and AluY resulting in the deletion of intervening sequences. Long-range PCR of the 5.5kb sequence in 494 normal lymphocyte samples showed heterozygous deletion in 28.3% of African- American ancestry samples but only in 4.8% of Caucasian samples (p<0.0001). This observation is strengthened by the copy number variation (CNV) data of the HapMap samples which showed that this deletion occurs in 27% of YRI (Yoruba – West African) population but none in non- African populations. The HapMap analysis further identified strong linkage disequilibrium between 5 single nucleotide polymorphisms and the 5.5kb deletion in the people of African ancestry. Computational analysis of 175kb sequence surrounding the deletion site revealed enhanced flexibility, low thermodynamic stability, high repetitiveness, and stable stem-loop/ hairpin secondary structures that are hallmarks of common fragile sites

Malaria Parasite CLAG3, a Protein Linked to Nutrient Channels, Participates in High Molecular Weight Membrane-Associated Complexes in the Infected Erythrocyte

<div><p>Malaria infected erythrocytes show increased permeability to a number of solutes important for parasite growth as mediated by the Plasmodial Surface Anion Channel (PSAC). The <i>P</i>. <i>falciparum clag3</i> genes have recently been identified as key determinants of PSAC, though exactly how they contribute to channel function and whether additional host/parasite proteins are required remain unknown. To begin to answer these questions, I have taken a biochemical approach. Here I have used an epitope-tagged CLAG3 parasite to perform co-immunoprecipitation experiments using membrane fractions of infected erythrocytes. Native PAGE and mass spectrometry studies reveal that CLAG3 participate in at least three different high molecular weight complexes: a ~720kDa complex consisting of CLAG3, RHOPH2 and RHOPH3; a ~620kDa complex consisting of CLAG3 and RHOPH2; and a ~480kDa complex composed solely of CLAG3. Importantly, these complexes can be found throughout the parasite lifecycle but are absent in untransfected controls. Extracellular biotin labeling and protease susceptibility studies localize the 480kDa complex to the erythrocyte membrane. This complex, likely composed of a homo-oligomer of 160kDa CLAG3, may represent a functional subunit, possibly the pore, of PSAC.</p></div

Solubilization and immunoprecipitation of membrane-associated CLAG3.

<p>(A) Western blot analysis shows that the zwitterionic detergents, lauryldimethyl amine oxide (LDAO) and fos-choline 12 (FC12), are able to solubilize integral CLAG3 at 0.5% (w/v) from sodium carbonate pH 11 (Na₂CO₃) treated trophozoite-infected erythrocyte membranes. (B) FC12 solubilizes total CLAG3 from (non-carbonate treated) membranes at concentrations 0.1% or above, leaving almost nothing behind in the insoluble membrane pellet. (LDS: lithium dodecyl sulfate). (C) Immunoprecipitation (IP) of CLAG3 from a previously described FLAG-tagged CLAG3 parasite (HB3^3REC) results in the appearance of the expected 160kDa CLAG3 band at FC12 concentrations ≥ 0.1%, and two additional faint bands (150kDa and 100kDa) at FC12 concentrations ≤ 0.05% [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157390#pone.0157390.ref008" target="_blank">8</a>]. (D) Substantially more CLAG3 remains in the detergent-soluble supernatant following a 16,000x<i>g</i> spin to pellet insoluble material as compared to a traditional 100,000x<i>g</i> spin for all detergents tested (SDOC: sodium deoxycholate). (E) IP experiments were repeated incorporating the slower 16,000x<i>g</i> spin. All detergents tested (with the exception of 0.5% FC12) give rise to the aforementioned three bands in non-carbonate (-Na₂CO₃) treated membranes, but fail to do so in membranes treated with sodium carbonate (+Na₂CO₃). As evidenced by the right half of each gel, the only detergent able to fully solubilize CLAG3 from membranes (and leave nothing behind in the insoluble pellet) is 0.5% FC12. The prominent 60kDa band corresponds to heavy chain IgG.</p

The 480kDa complex composed of CLAG3 localizes to the erythrocyte membrane.

<p>(A) Incubation of infected erythrocytes with extracellular NHS-LC-LC-Biotin results in labeling of intracellular proteins due to traversal through PSAC, which is prevented in the presence of PSAC inhibitor, MBX2366. (B) Membranes of biotin-labeled cells were solubilized with 1% FC12 and subjected to biotin IP, followed by Western blot for the indicated proteins. As indicated by the IP inputs, 1% FC12 is able to equally solubilize CLAG3, RHOPH2 and RHOPH3. However, only CLAG3 is found in the biotin IP eluate indicating localization to the erythrocyte surface. (C) Treatment with extracellular pronase-E does not result in the cleavage of intracellular proteins. (D) Extracellular pronase-E results in the appearance of a cleavage fragment for CLAG3, but not RHOPH2 and RHOPH3, in a dose-dependent fashion. C-terminal antibodies were used for all three proteins. (E) Extracellular biotin labeling results in labeling of the 480kDa complex in the presence of MBX2366 indicating that this complex localizes to the erythrocyte membrane. (F) Consistent with the above results, Western blot analysis with biotin antibody shows that only the CLAG3 obtained with 0.5% FC12 (corresponding to the 480kDa complex) labels with biotin in the presence of MBX2366.</p

CLAG3 forms high molecular weight membrane-associated complexes.

<p>(A) Blue Native PAGE analysis of FLAG IP eluates obtained from a FLAG-tagged CLAG3 parasite (HB3^3REC) and an isogenic untagged HB3 negative control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157390#pone.0157390.ref008" target="_blank">8</a>]. Two distinct bands can be seen in HB3^3REC that are absent in HB3: a ~720kDa band for concentrations of FC12 ≤ 0.05% and a ~480kDa band for concentrations of FC12 ≥ 0.1%. (B) FLAG Western blot confirms that the 720kDa and 480kDa bands contain CLAG3. (C) SDS PAGE analysis shows that upon addition of SDS and 2-mercaptoethanol the 720kDa complex dissociates to three proteins (CLAG3, RHOPH2, and RHOPH3 as determined by mass spectrometry), while the 480kDa complex dissociates to only one protein (CLAG3). (D) Sodium deoxycholate (SDOC) at 0.5% (w/v) yields an intermediate ~620kDa Native PAGE band that contains CLAG3 and RHOPH2, but not RHOPH3 (as determined by SDS PAGE and mass spectrometry). (E) A summary of the mass spectrometry results for the indicated Native and SDS PAGE bands. (F) IP experiments on tightly synchronized parasites show that CLAG3, RHOPH2 and RHOPH3 appear to associate faithfully in early (0–6 hours post-invasion), mid-stage (12–16 hours post-invasion), or late rings/early trophozoites (24–30 hours post-invasion). The prominent 30kDa band corresponds to light chain IgG.</p

Cross-linking studies with NHS-esters confirm the existence of CLAG3 complexes.

<p>(A) The properties of the three different N-hydroxysulfosuccinimide esters (NHS-esters) used in this study: Bis(sulfosuccinimidyl) suberate (BS3), Disuccinimidyl glutarate (DSG), and Dithiobis (succinimidyl propionate) (DSP). (B) The 480kDa complex is observed with the use of 1% FC12, whereas addition of the three different NHS-esters results in the appearance of higher molecular weight complexes in a dose dependent manner (as determined by Native PAGE). (C) DSP cross-link was reversed with the addition of 100mM Dithiothreitol (DTT) and bands separated on SDS PAGE. The three prominent bands observed are consistent in size with CLAG3, RHOPH2, and RHOPH3. The band near 250kDa was subsequently found to also be present in HB3 negative control parasites (see next panel). (D) IP experiments on tightly synchronized parasites obtained without enrichment and cross-linked with DSP demonstrate no appreciable differences in the association CLAG3, RHOPH2, and RHOPH3 in early (0–12 hour) or late (12–24 hour) rings (when PSAC is inactive and active, respectively). HB3 was used as a negative control.</p

SIRT1 is a positive regulator of the master osteoblast transcription factor, RUNX2

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Activation of SIRT1 has previously been shown to protect mice against osteoporosis through yet ill-defined mechanisms. In this study, we outline a role for SIRT1 as a positive regulator of the master osteoblast transcription factor, RUNX2. We find that ex vivo deletion of sirt1 leads to decreased expression of runx2 downstream targets, but not runx2 itself, along with reduced osteoblast differentiation. Reciprocally, treatment with a SIRT1 agonist promotes osteoblast differentiation, as well as the expression of runx2 downstream targets, in a SIRT1-dependent manner. Biochemical and luciferase reporter assays demonstrate that SIRT1 interacts with and promotes the transactivation potential of RUNX2. Intriguingly, mice treated with the SIRT1 agonist, resveratrol, show similar increases in the expression of RUNX2 targets in their calvaria (bone tissue), validating SIRT1 as a physiologically relevant regulator of RUNX2