Immature natural killer cells promote progression of triple-negative breast cancer.

Natural killer (NK) cells are cytotoxic lymphocytes that accumulate within the tumor microenvironment and are generally considered to be antitumorigenic. Using single-cell RNA sequencing and functional analysis of multiple triple-negative breast cancer (TNBC) and basal tumor samples, we observed a unique subcluster of Socs3highCD11b-CD27- immature NK cells that were present only in TNBC samples. These tumor-infiltrating NK cells expressed a reduced cytotoxic granzyme signature and, in mice, were responsible for activating cancer stem cells through Wnt signaling. NK cell-mediated activation of these cancer stem cells subsequently enhanced tumor progression in mice, whereas depletion of NK cells or Wnt ligand secretion from NK cells by LGK-974 decreased tumor progression. In addition, NK cell depletion or inhibition of their function improved anti-programmed cell death ligand 1 (PD-L1) antibody or chemotherapy response in mice with TNBC. Furthermore, tumor samples from patients with TNBC and non-TNBC revealed that increased numbers of CD56bright NK cells were present in TNBC tumors and were correlated to poor overall survival in patients with TNBC. Together, our findings identify a population of protumorigenic NK cells that may be exploited for both diagnostic and therapeutic strategies to improve outcomes for patients with TNBC.

Ormeloxifene, a nonsteroidal antifertility drug promotes megakaryocyte differentiation in leukemia cell line K562.

Ormeloxifene (ORM) (3,4-trans-2,2-dimethyl-3-phenyl-4-p-(ß-pyrrolidinoethoxy) phenyl-7-methoxychroman), world's first nonsteroidal selective estrogen receptor modulator approved for contraception in India has been shown to have potential anticancer activities. Here, we show that ORM can induce megakaryocyte and myeloid (granulocytic) but not erythroid differentiation in multipotent human myeloid leukemia cell line K562. We show that ORM at an IC50 of 7.5 µM can induce morphological changes similar to megakaryocytes in K562 cells. ORM led to increase in levels of megakaryocytic differentiation markers (CD41 and CD61) as well as key transcription factors GATA1 and AML1. We further show that ORM induces megakaryocytic differentiation in K562 cells through ERK activation and induction of autophagy in a fashion similar to other known inducers of megakaryocytic differentiation such as phorbol esters. In addition, as shown earlier, we yet again observed that ORM led to activation of caspases since their inhibition through pan-caspase inhibitor mitigated megakaryocytic differentiation as they led to significant decrease in CD41 and CD61. Because induction of megakaryocytic differentiation in K562 involves growth arrest and exit from cell cycle, we also observed an increase in levels of p21 and p27 with decrease in c-Myc protein levels in K562 cells treated with 7.5 µM ORM for 24 and 48 h, respectively. Taken together, these findings indicate that ORM can markedly induce megakaryocytic differentiation in K562 cells.

Dll1-Mediated Notch Signaling Drives Tumor Cell Cross-talk with Cancer-Associated Fibroblasts to Promote Radioresistance in Breast Cancer.

SIGNIFICANCE: Dll1+ breast cancer cells activate Notch signaling in cancer-associated fibroblasts that increases Wnt ligand secretion and leads to ß-catenin-driven radioresistance and metastasis, opening new therapeutic avenues for breast cancer.

SOX4-mediated FBW7 transcriptional upregulation confers Tamoxifen resistance in ER+ breast cancers via GATA3 downregulation.

AIM: Tamoxifen-mediated endocrine therapy has been standard treatment for ER+ breast cancers; however, majority of them acquire resistance leading to disease relapse. Although numerous substrates of E3 ligase FBW7 are known, only a handful of factors that regulate FBW7 expression and function are reported. In particular, there remains a lack of in-depth understanding of FBW7 transcriptional regulation. MATERIALS AND METHODS: Luciferase reporter assay was performed after cloning full length and truncated FBW7 promoters followed by Chromatin immunoprecipitation assay to validate binding of SOX4 on FBW7 promoter. Transcriptional regulation of FBW7 by SOX4 and their biological consequences with respect to ER+ breast cancer was then evaluated using immunoblotting and other cell based assays. KEY FINDINGS: SOX4 positively regulates FBW7 at transcriptional level by binding to three putative SOX4 biding sites within 3.1 kb long FBW7 promoter. Analysis of publicly available RNAseq datasets also showed a positive correlation between SOX4 and FBW7 mRNA in cancer cell lines and patient samples. qPCR and Immunoblotting confirmed that transiently or stably expressed SOX4 induced both endogenous FBW7 mRNA and protein levels. Our findings further demonstrated that increased levels of SOX4 and FBW7 in MCF7 mammospheres promoted cancer stemness and tumor cell dormancy. We further showed that both MCF7 mammospheres and MCFTAMR cells had elevated SOX4 levels which apparently enhanced FBW7 to potentiate GATA3 degradation leading to enhanced stemness, tumor dormancy and Tamoxifen resistance in MCF7TAMR as well as patients with ER+ breast cancers. SIGNIFICANCE: Targeting SOX4-FBW7-GATA3 axis may overcome tamoxifen resistance in ER+ breast cancers.

Proteomic analysis of TGFß-induced A549 secretome identifies putative regulators of epithelial-mesenchymal transition.

Imparting epithelial to mesenchymal transition (EMT) during cellular transformation, a major driving force behind tumor progression, is one of the notorious oncogenic activities of transforming growth factor ß (TGFß); however, the secretary factors released during TGFß-induced EMT that may have role in potentiating EMT and tumor progression are poorly known. This study was undertaken to identify such secreted protein factors from TGFß-induced A549 cells cultured in serum-free chemically defined medium (FreestyleTM ) using Matrix Assisted Laser Desorption Ionization-Time of flight/Time of flight (MALDI-TOF/TOF) mass spectrometry. We identified some of the potential factors such as ESR, ANXA2, ALDH1A, TGFß-induced protein ig-h3, and PAI-1 that were not only secreted but some were also elevated in TGFß-induced A549 cells. Interestingly, these factors are widely reported to play crucial role in EMT induction and progression, which not only validates our findings but also opens avenues for further investigation, if upon secretion they act exogenously through certain receptors to potentiate cellular signaling involved in EMT induction and tumor progression.

CDK2-instigates C/EBPα degradation through SKP2 in Acute myeloid leukemia.

Transcription factor CCAAT/enhancer-binding protein-alpha (C/EBPα) regulates myelopoiesis by coupling growth arrest with differentiation of myeloid progenitors. Mutations in one or both alleles are observed in 10-14% AML cases that render C/EBPα functionally inactive. Besides, antagonistic protein-protein interactions also impair C/EBPα expression and function. In recent independent studies, we showed that CDK2 and SKP2 downregulated C/EBPα expression in an ubiquitin-dependent proteasome degradation manner leading to differentiation block in AML. Here, we demonstrate that CDK2-instigated C/EBPα downregulation is actually mediated by SKP2. Mechanistically, we show that CDK2 stabilizes SKP2 by phosphorylating it at Ser64 and thereby potentiates C/EBPα ubiquitination and subsequent degradation in AML cells. Immunoblot experiments showed that CDK2 inhibition downregulated SKP2 levels and concomitantly enhanced C/EBPα levels in myeloid cells. We further show that while CDK2 promoted C/EBPα ubiquitination and inhibited its protein levels, negatively affected its transactivation potential and DNA binding ability, simultaneous SKP2 depletion abrogated CDK2-promoted ubiquitination and restored C/EBPα expression and function. Taken together, these findings consolidate that CDK2 potentiates SKP2-mediated C/EBPα degradation in AML and targeting CDK2-SKP2 axis can be harnessed for therapeutic benefit in AML. Hypothetical model depicts that SKP2-mediated C/EBPα proteasomal degradation is reinforced by CDK2. CDK2 phopshorylates SKP2 leading to its enhanced stabilization which in turn exaggerates C/EBPα degradation leading to differentiation arrest in AML.

FBW7 Inhibits Myeloid Differentiation in Acute Myeloid Leukemia via GSK3-Dependent Ubiquitination of PU.1.

Glycogen synthase kinase 3ß (GSK3ß), an ubiquitously expressed serine/threonine kinase is reported to be overexpressed and hyperactivated in cancers including acute myeloid leukemia (AML) where it promotes self-renewal, growth, and survival of AML cells. Therefore, GSK3ß inhibition results in AML cell growth inhibition and myeloid differentiation. Here we identified master transcription factor PU.1 of monocyte-macrophage differentiation pathway as potential GSK3ß target. We demonstrate that GSK3ß phosphorylates PU.1 at Ser41 and Ser140 leading to its recognition and subsequent ubiquitin-mediated degradation by E3 ubiquitin ligase FBW7. This GSK3-dependent degradation of PU.1 by FBW7 inhibited monocyte-macrophage differentiation. We further showed that a phospho-deficient PU.1 mutant (PU.1-S41, S140A) neither bound to FBW7 nor was degraded by it. Consequently, PU.1-S41, S140A retained its transactivation, DNA-binding ability and promoted monocyte-macrophage differentiation of U937 cells even without phorbol 12-myristate 13-acetate (PMA) treatment. We further showed that FBW7 overexpression inhibited both PMA as well as M-CSF-induced macrophage differentiation of myeloid cell lines and peripheral blood mononuclear cells (PBMC) from healthy volunteers, respectively. Contrarily, FBW7 depletion promoted differentiation of these cells even without any inducer suggesting for a robust role of GSK3ß-FBW7 axis in negatively regulating myeloid differentiation. Furthermore, we also recapitulated these findings in PBMCs isolated from patients with leukemia where FBW7 overexpression markedly inhibited endogenous PU.1 protein levels. In addition, PBMCs also showed enhanced differentiation when treated with M-CSF and GSK3 inhibitor (SB216763) together compared with M-CSF treatment alone. IMPLICATIONS: Our data demonstrate a plausible mechanism behind PU.1 restoration and induction of myeloid differentiation upon GSK3ß inhibition and further substantiates potential of GSK3ß as a therapeutic target in AML.

Chebulinic acid inhibits MDA-MB-231 breast cancer metastasis and promotes cell death through down regulation of SOD1 and induction of autophagy.

Triple-negative breast cancers (TNBC) are highly aggressive and drug resistant accounting for majority of cases with poor outcome. Purified natural compounds display substantial anticancer activity with reduced cytotoxicity providing a new avenue to combat TNBC. Chebulinic acid (CA), a polyphenol derived from the fruits of various medicinal plants has potent anticancer activity. Here, we demonstrate that CA shows significant cytotoxicity against triple negative MDA-MB-231 cells. CA exhibited cytotoxicity to MDA-MB-231 cells in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Further, CA mitigated MDA-MB-231 cells viability and proliferation as shown by reduced live cell count, crystal violet staining, colony formation assay, soft agar assay and cell cycle analysis. Wound healing assay and trans-well migration assay demonstrated that CA significantly inhibited migration of MDA-MB-231 cells. Also reduced MMP9 expression was observed in CA-treated cells by gelatin zymography. CA negatively regulated mesenchymal characteristics of MDA-MB-231 cells demonstrated by F-actin staining and reduced expression of N-cadherin by confocal microscopy and western blot analysis. Annexin V/propidium iodide (PI) and active caspase-3 staining showed that CA was able to induce apoptosis in MDA-MB-231 cells but did not activate caspase-3. Two-dimensional gel electrophoresis based proteomic analysis demonstrated that CA regulated proteins belonging to the oxidative stress pathway, apoptotic pathway and proteins with antiproliferative activity. Western blot analysis analysis revealed that CA negatively regulated superoxide dismutase 1 (SOD1) and enhanced oxidative stress in MDA-MB-231 cells. SOD1 in-gel activity assay also showed reduced SOD1 activity upon CA treatment. Overexpression studies with GFP-LC3 and tandem tagged RFP-GFP-LC-3 also demonstrated enhanced autophagy upon CA treatment.

E3 ligase SCFSKP2 ubiquitinates and degrades tumor suppressor C/EBPα in acute myeloid leukemia.

AIM: Transcription factor CCAAT/Enhancer binding protein alpha (C/EBPα) is a key regulator of myeloid differentiation, granulopoiesis in particular. Although CEBPA mutations are found in more than 10% in AML, functional inhibition of C/EBPα protein is also widely observed in AML. Here, we sought to examine if SKP2, an aberrantly enhanced E3 ubiquitin ligase in primary AMLs inhibits C/EBPα stability to induce differentiation block. MAIN METHODS: Here we employed cell based assays such as transfections, immunoblotting, co-immunoprecipitation, luciferase and gel shift assays along with differentiation assays to investigate SKP2 regulated C/EBPα protein stability in acute myeloid leukemia. KEY FINDINGS: Here we discovered that oncogenic E3 ubiquitin ligase SCFskp2 ubiquitinates and destabilizes C/EBPα in a proteasome-dependent manner. Our data demonstrates that SKP2 physically interacts with C-terminal of C/EBPα and promotes its K48-linked ubiquitination-mediated degradation leading to its reduced transactivation potential, DNA binding ability and cellular functions. We further show that while overexpression of SKP2 inhibits both ectopic as well as endogenous C/EBPα in heterologous (HEK293T) as well as myeloid leukemia cells respectively, SKP2 depletion restores endogenous C/EBPα leading to reduced colony formation and enhanced myeloid differentiation of myeloid leukemia cells. Using Estradiol-inducible K562-C/EBPα-ER cells as yet another model of granulocytic differentiation, we further confirmed that SKP2 overexpression indeed inhibits granulocytic differentiation by mitigating C/EBPα stability. SIGNIFICANCE: Our findings identify SKP2 as a potential negative regulator of C/EBPα stability and function in AML which suggests that SKP2 can be potentially targeted in AML to restore C/EBPα and overcome differentiation block.

Loss of ELF5-FBXW7 stabilizes IFNGR1 to promote the growth and metastasis of triple-negative breast cancer through interferon-γ signalling.

Triple-negative breast cancer (TNBC) is characterized by a high degree of immune infiltrate in the tumour microenvironment, which may influence the fate of TNBC cells. We reveal that loss of the tumour suppressive transcription factor Elf5 in TNBC cells activates intrinsic interferon-γ (IFN-γ) signalling, promoting tumour progression and metastasis. Mechanistically, we find that loss of the Elf5-regulated ubiquitin ligase FBXW7 ensures stabilization of its putative protein substrate IFN-γ receptor 1 (IFNGR1) at the protein level in TNBC. Elf5low tumours show enhanced IFN-γ signalling accompanied by an increase of immunosuppressive neutrophils within the tumour microenvironment and increased programmed death ligand 1 expression. Inactivation of either programmed death ligand 1 or IFNGR1 elicited a robust anti-tumour and/or anti-metastatic effect. A positive correlation between ELF5 and FBXW7 expression and a negative correlation between ELF5, FBXW7 and IFNGR1 expression in the tumours of patients with TNBC strongly suggest that this signalling axis could be exploited for patient stratification and immunotherapeutic treatment strategies for Elf5low patients with TNBC.

CDK2 destabilizes tumor suppressor C/EBPα expression through ubiquitin-mediated proteasome degradation in acute myeloid leukemia.

Deregulation and functional inhibition of CCAAT-enhancer-binding protein α (C/EBPα), a key transcription factor of myeloid lineage leads to development of myeloid leukemia. In this study, we show that cyclin-dependent kinase 2 (CDK2) negatively regulates C/EBPα protein levels in myeloid leukemia cells. The overexpression of CDK2 inhibited C/EBPα both in a heterologous HEK293T and U937 myeloid leukemia cells. On the contrary, CDK2 depletion enhanced endogenous C/EBPα protein levels. CDK2 mitigated C/EBPα levels by promoting its ubiquitin-mediated proteasome degradation. We further showed that although CDK2 interacted with C/EBPα, direct interaction of CDK2 with C/EBPα is not involved in C/EBPα downregulation. CDK2-dependent phosphorylation of C/EBPα on its widely reported phosphorylatable amino acid residues is apparently not required for C/EBPα degradation by CDK2. Furthermore, our data demonstrate that CDK2-driven C/EBPα inhibition mitigates its transactivation potential and cellular functions such as ability to promote myeloid differentiation and growth arrest.

Nano-LC based proteomic approach identifies that E6AP interacts with ENO1 and targets it for degradation in breast cancer cells.

E6AP (E6 associated protein) is a HECT domain containing protein having dual E3 ligase and ERα coactivation activity in breast cancer cells. Although E6AP is known to possess antitumorigenic activity, the underlying molecular mechanism is poorly understood. In the present study, we applied nano-LC based proteomics approach to identify E6AP-interacting proteins where we performed GST-pull down using GST-E6AP from whole cell extracts of MCF7 cells, resolved the differentially interacting proteins on 1D-SDS-PAGE, excised the gel bands that were trypsin digested followed by fractionation and spotting on MALDI-TOF/TOF plate through Nano-LC MALDI spotter. Subsequently, fractionated and spotted peptides were identified using MALDI-TOF/TOF. We identified several E6AP interacting proteins including previously reported such as HSP70 and new ones such as Enolase-1. We further confirmed that E6AP and Enolase1 interacted and colocalized more in the cytoplasmic periphery in breast cancer cells and further demonstrated that E6AP also targeted ENO1 for ubiquitin-mediated degradation in these cells.

BMP signaling-driven osteogenesis is critically dependent on Prdx-1 expression-mediated maintenance of chondrocyte prehypetrophy.

During endochondral ossification, cartilage template is eventually replaced by bone. This process involves several well characterized, stereotypic, molecular and cellular changes in the cartilage primordia. These steps involve transition from resting to proliferative and then pre-hypertrophic to finally hypertrophic cartilage. BMP signaling is necessary and sufficient for osteogenesis. However, the specific step(s) of endochondral ossification in which BMP signaling plays an essential role is not yet known. In this study we have identified Prdx1, a known scavenger of ROS, to be expressed in pre-hypertrophic chondrocytes in a BMP signaling-dependent manner. We demonstrate that BMP signaling inhibition increases ROS levels in osteogenic cells. Further, Prdx1 regulates osteogenesis in vivo by helping maintenance of Ihh expressing pre-hypertrophic cells, in turn regulating these cells' transition into hypertrophy. Therefore, our data suggests that one of the key roles of BMP signaling in endochondral ossification is to maintain pre-hypertrophic state.

Chebulinic Acid Isolated From the Fruits of Terminalia chebula Specifically Induces Apoptosis in Acute Myeloid Leukemia Cells.

Chebulinic acid, an ellagitannin found in the fruits of Terminalia chebula, has been extensively used in traditional Indian system of medicine. It has shown to have various biological activities including antitumor activity. The present study aims to investigate the cytotoxic potential of chebulinic acid in human myeloid leukemia cells. Interestingly, chebulinic acid caused apoptosis of acute promyelocytic leukemia HL-60 and NB4 cells but not K562 cells. In vitro antitumor effects of chebulinic acid were investigated by using various acute myeloid leukemia cell lines. Chebulinic acid treatment to HL-60 and NB4 cells induced caspase activation, cleavage of poly(ADP-ribose) polymerase, DNA fragmentation, chromatin condensation, and changes in the mitochondrial membrane permeability. Additionally, inhibition of caspase activation drastically reduced the chebulinic acid-induced apoptosis of acute promyelocytic leukemia cells. Our data also demonstrate that chebulinic acid-induced apoptosis in HL-60 and NB4 cells involves activation of extracellular signal-regulated kinases, which, when inhibited with ERK inhibitor PD98059, mitigates the chebulinic acid-induced apoptosis. Taken together, our findings exhibit the selective potentiation of chebulinic acid-induced apoptosis in acute promyelocytic leukemia cells. Copyright © 2017 John Wiley & Sons, Ltd.

E6AP inhibits G-CSFR turnover and functions by promoting its ubiquitin-dependent proteasome degradation.

Granulocyte colony-stimulating factor receptor (G-CSFR) plays a crucial role in regulating myeloid cell survival, proliferation, and neutrophilic granulocyte precursor cells maturation. Previously, we demonstrated that Fbw7α negatively regulates G-CSFR and its downstream signaling through ubiquitin-proteasome mediated degradation. However, whether additional ubiquitin ligases for G-CSFR exist is not known. Identifying multiple E3 ubiquitin ligases for G-CSFR shall improve our understanding of activation and subsequent attenuation of G-CSFR signaling required for differentiation and proliferation. Here, for the first time we demonstrate that E6 associated protein (E6AP), an E3 ubiquitin ligase physically associates with G-CSFR and targets it for ubiquitin-mediated proteasome degradation and thereby attenuates its functions. We further show that E6AP promoted G-CSFR degradation leads to reduced phosphorylation of signal transducer and activator of transcription 3 (STAT3) which is required for G-CSF dependent granulocytic differentiation. More importantly, our finding shows that E6AP also targets mutant form of G-SCFR (G-CSFR-T718), frequently observed in severe congenital neutropenia (SCN) patients that very often culminate to AML, however, at a quite slower rate than wild type G-CSFR. In addition, our data showed that knockdown of E6AP restores G-CSFR and its signaling thereby promoting granulocytic differentiation. Collectively, our data demonstrates that E6AP facilitates ubiquitination and subsequent degradation of G-CSFR leading to attenuation of its downstream signaling and inhibition of granulocytic differentiation.

Ubiquitin Ligase, Fbw7, Targets CDX2 for Degradation via Two Phosphodegron Motifs in a GSK3ß-Dependent Manner.

Drosophila caudal-related homeobox transcription factor 2 (CDX2) drives differentiation of the intestinal epithelium. Loss of CDX2 expression has been reported in several colorectal cancers and cancer cell lines with a potential inverse correlation between CDX2 levels and tumor stage. Ubiquitination of CDX2 leading to its downregulation has been implicated in several studies; however, the E3 ubiquitin ligases involved in CDX2 ubiquitination have largely remained unknown. Here, it is mechanistically determined that the E3 ubiquitin ligase Fbw7 promotes CDX2 ubiquitination and degradation through two phosphodegron motifs present within CDX2 in a GSK3ß-dependent manner leading to its reduced expression and function in colon cancer cells. Fbw7, through its WD domain, interacted with CDX2 both in a heterologous HEK293T cell system and in colon cancer cells. GSK3ß was also present in the same complex as determined by coimmunoprecipitation. Furthermore, overexpression of both Fbw7 or GSK3ß down regulated endogenous CDX2 expression and function; however, both failed to inhibit endogenous CDX2 when either of them were depleted in colon cancer cells. Fbw7-mediated inhibition of CDX2 expression also led to reduced CDX2 transactivation and growth arrest of colon cancer cells. Both GSK3ß and Fbw7 degraded mutant-CDX2 having either of the Cdc4-phosphodegron (CPD) motifs disrupted (CDX2-S60A or CDX-S281A), but were unable to degrade mutant-CDX2 having both CPDs disrupted (CDX2-S60,64,281A). IMPLICATIONS: Taken together, these findings demonstrate that Fbw7 negatively regulates CDX2 expression in a GSK3ß-dependent manner through two CPDs present in CDX2. Mol Cancer Res; 14(11); 1097-109. ©2016 AACR.

Skp2 inhibits osteogenesis by promoting ubiquitin-proteasome degradation of Runx2.

Osteogenic transcription factor Runx2 is essential for osteoblast differentiation. The activity of Runx2 is tightly regulated at transcriptional as well as post-translational level. However, regulation of Runx2 stability by ubiquitin mediated proteasomal degradation by E3 ubiquitin ligases is little-known. Here, for the first time we demonstrate that Skp2, an SCF family E3 ubiquitin ligase negatively targets Runx2 by promoting its polyubiquitination and proteasome dependent degradation. Co-immunoprecipitation studies revealed that Skp2 physically interacts with Runx2 both in a heterologous as well as physiologically relevant system. Functional consequences of Runx2-Skp2 physical interaction were then assessed by promoter reporter assay. We show that Skp2-mediated downregulation of Runx2 led to reduced Runx2 transactivation and osteoblast differentiation. On the contrary, inhibition of Skp2 restored Runx2 levels and promoted osteoblast differentiation. We further show that Skp2 and Runx2 proteins are co-expressed and show inverse relation in vivo such as in lactating, ovariectomized and estrogen-treated ovariectomized animals. Together, these data demonstrate that Skp2 targets Runx2 for ubiquitin mediated degradation and hence negatively regulate osteogenesis. Therefore, the present study provides a plausible therapeutic target for osteoporosis or cleidocranial dysplasia caused by the heterozygous mutation of Runx2 gene.

E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation.

Runx2, a master regulator of osteoblast differentiation, is tightly regulated at both transcriptional and post-translational levels. Post-translational modifications such as phosphorylation and ubiquitination have differential effects on Runx2 functions. Here, we show that the reduced expression and functions of Runx2 upon its phosphorylation by GSK3ß are mediated by its ubiquitin-mediated degradation through E3 ubiquitin ligase Fbw7α. Fbw7α through its WD domain interacts with Runx2 both in a heterologous (HEK293T cells) system as well as in osteoblasts. GSK3ß was also present in the same complex as determined by co-immunoprecipitation. Furthermore, overexpression of either Fbw7α or GSK3ß was sufficient to down-regulate endogenous Runx2 expression and function; however, both failed to inhibit endogenous Runx2 when either of them was depleted in osteoblasts. Fbw7α-mediated inhibition of Runx2 expression also led to reduced Runx2 transactivation and osteoblast differentiation. In contrast, inhibition of Fbw7α restored Runx2 levels and promoted osteoblast differentiation. We also observed reciprocal expression levels of Runx2 and Fbw7α in models of bone loss such as lactating (physiological bone loss condition) and ovariectomized (induction of surgical menopause) animals that show reduced Runx2 and enhanced Fbw7α, whereas this was reversed in the estrogen-treated ovariectomized animals. In addition, methylprednisolone (a synthetic glucocorticoid) treatment to neonatal rats showed a temporal decrease in Runx2 with a reciprocal increase in Fbw7 in their calvarium. Taken together, these data demonstrate that Fbw7α negatively regulates osteogenesis by targeting Runx2 for ubiquitin-mediated degradation in a GSK3ß-dependent manner and thus provides a plausible explanation for GSK3ß-mediated bone loss as described before.