Uma Sankar

Microphthalmia Transcription Factor Is a Target of the p38 MAPK Pathway in Response to Receptor Activator of NF-κB Ligand Signaling

Receptor activator of NF-κB ligand (RANKL) activates signaling pathways that regulate osteoclast differentiation, function, and survival. The microphthalmia transcription factor (MITF) is required for terminal differentiation of osteoclasts. To determine whether MITF could be a target of RANKL signaling, a phosphospecific MITF antibody directed against conserved residue Ser307, a potential mitogen-activated protein kinase (MAPK) site, was produced. Using this antibody, we could demonstrate that MITF was rapidly and persistently phosphorylated upon stimulation of primary osteoclasts with RANKL and that phosphorylation of Ser307 correlated with expression of the target gene tartrate-resistant acid phosphatase. MITF phosphorylation at Ser307 also correlated with persistent activation of p38 MAPK, and p38 MAPK could utilize MITF Ser307 as a substratein vitro. The phosphorylation of MITF and activation of target gene expression in osteoclasts were blocked by p38 MAPK inhibitor SB203580. In transient transfections, a constitutively active Rac1 or MKK6 gene could collaborate with MITF to activate the tartrate-resistant acid phosphatase gene promoter dependent on Ser307. Dominant negative p38 α and β could inhibit the collaboration between upstream signaling components and MITF in the transient assays. These results indicate that MITF is a target for the RANKL signaling pathway in osteoclasts and that phosphorylation of MITF leads to an increase in osteoclast-specific gene expression.

Identification of Adiponectin as a Novel Hemopoietic Stem Cell Growth Factor

The hemopoietic microenvironment consists of a diverse repertoire of cells capable of providing signals that influence hemopoietic stem cell function. Although the role of osteoblasts and vascular endothelial cells has recently been characterized, the function of the most abundant cell type in the bone marrow, the adipocyte, is less defined. Given the emergence of a growing number of adipokines, it is possible that these factors may also play a role in regulating hematopoiesis. Here, we investigated the role of adiponectin, a secreted molecule derived from adipocytes, in hemopoietic stem cell (HSC) function. We show that adiponectin is expressed by components of the HSC niche and its’ receptors AdipoR1 and AdipoR2 are expressed by HSCs. At a functional level, adiponectin influences HSCs by increasing their proliferation, while retaining the cells in a functionally immature state as determined by in vitro and in vivo assays. We also demonstrate that adiponectin signaling is required for optimal HSC proliferation both in vitro and in long term hemopoietic reconstitution in vivo. Finally we show that adiponectin stimulation activates p38 MAPK, and that inhibition of this pathway abrogates adiponectin’s proliferative effect on HSCs. These studies collectively identify adiponectin as a novel regulator of HSC function and suggest that it acts through a p38 dependent pathway.

Growth factor erv1-like modulates Drp1 to preserve mitochondrial dynamics and function in mouse embryonic stem cells.

Gfer links mitochondrial dynamics with pluripotency in mouse embryonic stem cells through its modulation of the mitochondrial fission GTPase Drp1.

Eos, MITF, and PU.1 Recruit Corepressors to Osteoclast-Specific Genes in Committed Myeloid Progenitors

ABSTRACT Transcription factors MITF and PU.1 collaborate to increase expression of target genes like cathepsin K (Ctsk) and acid phosphatase 5 (Acp5) during osteoclast differentiation. We show that these factors can also repress transcription of target genes in committed myeloid precursors capable of forming either macrophages or osteoclasts. The direct interaction of MITF and PU.1 with the zinc finger protein Eos, an Ikaros family member, was necessary for repression of Ctsk and Acp5. Eos formed a complex with MITF and PU.1 at target gene promoters and suppressed transcription through recruitment of corepressors CtBP (C-terminal binding protein) and Sin3A, but during osteoclast differentiation, Eos association with Ctsk and Acp5 promoters was significantly decreased. Subsequently, MITF and PU.1 recruited coactivators to these target genes, resulting in robust expression of target genes. Overexpression of Eos in bone marrow-derived precursors disrupted osteoclast differentiation and selectively repressed transcription of MITF/PU.1 targets, while small interfering RNA knockdown of Eos resulted in increased basal expression of Ctsk and Acp5. This work provides a mechanism to account for the modulation of MITF and PU.1 activity in committed myeloid progenitors prior to the initiation of osteoclast differentiation in response to the appropriate extracellular signals.

RANKL coordinates cell cycle withdrawal and differentiation in osteoclasts through the cyclin-dependent kinase inhibitors p27KIP1 and p21CIP1.

The coordination of cell cycle progression and osteoclast differentiation by RANKL signaling was studied. Experiments with mouse genetic models revealed that RANKL promoted cell cycle withdrawal of osteoclast precursors dependent on the cyclin kinase inhibitor p27‐KIP1, but that both p27‐KIP1 and p21‐CIP1 were required for osteoclast differentiation. These cyclin inhibitors may directly regulate osteoclast differentiation in addition to regulating cell cycle withdrawal.

Mitochondria: A sulfhydryl oxidase and fission GTPase connect mitochondrial dynamics with pluripotency in embryonic stem cells

Mitochondria have long been recognized as cellular energy power houses that also regulate cellular redox signaling to arbitrate cell survival. Recent studies of mitochondria in stem cells (SCs) demonstrate that they have critical roles beyond this traditional view. Embryonic (E) SCs, termed pluripotent for their ability to differentiate into all cell types within an organism, maintain a limited number of morphologically undifferentiated (electron translucent and poorly formed cristae) mitochondria. As these cells differentiate, their mitochondria undergo a tightly choreographed gain of number, mass and morphological complexity. Therefore, mechanisms that regulate mitochondrial growth, localization, division and partition must play active roles in the maintenance of pluripotency and execution of differentiation. Aberrant mitochondrial dynamics are associated with a plethora of human disorders, for which SCs hold curative potential. Hence, a comprehensive understanding of the mechanisms that regulate mitochondrial dynamics and function in SCs and their overall relationship to the maintenance of pluripotency is pivotal for the progression of therapeutic regenerative medicine.

Inhibition of Ca2+/Calmodulin–Dependent Protein Kinase Kinase 2 Stimulates Osteoblast Formation and Inhibits Osteoclast Differentiation

Bone remodeling, a physiological process characterized by bone formation by osteoblasts (OBs) and resorption of preexisting bone matrix by osteoclasts (OCs), is vital for the maintenance of healthy bone tissue in adult humans. Imbalances in this vital process result in pathological conditions including osteoporosis. Owing to its initial asymptomatic nature, osteoporosis is often detected only after the patient has sustained significant bone loss or a fracture. Hence, anabolic therapeutics that stimulate bone accrual is in high clinical demand. Here we identify Ca2+/calmodulin (CaM)‐dependent protein kinase kinase 2 (CaMKK2) as a potential target for such therapeutics because its inhibition enhances OB differentiation and bone growth and suppresses OC differentiation. Mice null for CaMKK2 possess higher trabecular bone mass in their long bones, along with significantly more OBs and fewer multinuclear OCs. In vitro, although Camkk2−/− mesenchymal stem cells (MSCs) yield significantly higher numbers of OBs, bone marrow cells from Camkk2−/− mice produce fewer multinuclear OCs. Acute inhibition of CaMKK2 by its selective, cell‐permeable pharmacological inhibitor STO‐609 also results in increased OB and diminished OC formation. Further, we find phospho‐protein kinase A (PKA) and Ser133 phosphorylated form of cyclic adenosine monophosphate (cAMP) response element binding protein (pCREB) to be markedly elevated in OB progenitors deficient in CaMKK2. On the other hand, genetic ablation of CaMKK2 or its pharmacological inhibition in OC progenitors results in reduced pCREB as well as significantly reduced levels of its transcriptional target, nuclear factor of activated T cells, cytoplasmic (NFATc1). Moreover, in vivo administration of STO‐609 results in increased OBs and diminished OCs, conferring significant protection from ovariectomy (OVX)‐induced osteoporosis in adult mice. Overall, our findings reveal a novel function for CaMKK2 in bone remodeling and highlight the potential for its therapeutic inhibition as a valuable bone anabolic strategy that also inhibits OC differentiation in the treatment of osteoporosis.

Gfer Inhibits Jab1-Mediated Degradation of p27kip1 to Restrict Proliferation of Hematopoietic Stem Cells

Genes that promote hematopoietic stem cell (HSC) quiescence play important roles in the maintenance of their function. Here we show a novel role for the evolutionarily conserved flavin adenine dinucleotide (FAD)-dependent sulfhydryl oxidase, growth factor erv1-like (Gfer) in the restriction of HSC proliferation through its inhibition of Jab1-mediated turnover of p27kip1.

Novel genes regulated by microphthalmia transcription factor in macrophages and osteoclasts

S343 A bsracts age 45 and 18.5% reported a fall resulting in a fracture within the past 5 years. The mean (SD) serum 25(OH)D was 30.4 (13.2) ng/mL, median 29 ng/mL (range 7-212 ng/mL). 1.1% had 25(OH)D <9 ng/mL, 8.1% < 15 ng/mL, 18% < 20 ng/mL, 36% < 25 ng/mL and 52% < 30 ng/mL. 16.5% had evidence of secondary hyperparathyroidism as defined by a normal serum calcium and an elevated PTH. The prevalence of D insufficiency (25(OH)D < 25 ng/mL) was significantly higher in subjects who took less than 400 IU of D supplementation daily compared with those who took at least 400 IU daily (47% vs 28% respectively, p<0.001). This study demonstrates that approximately 50% of North American women currently receiving treatment for osteoporosis have suboptimal 25(OH)D levels. Despite routine recommendations for OTC vitamin D supplementation by physicians, D insufficiency is highly prevalent even among women diagnosed and treated for osteoporosis.

A novel crosstalk between calcium/calmodulin kinases II and IV regulates cell proliferation in myeloid leukemia cells

CaMKs link transient increases in intracellular Ca(2+) with biological processes. In myeloid leukemia cells, CaMKII, activated by the bcr-abl oncogene, promotes cell proliferation. Inhibition of CaMKII activity restricts cell proliferation, and correlates with growth arrest and differentiation. The mechanism by which the inhibition of CaMKII results in growth arrest and differentiation in myeloid leukemia cells is still unknown. We report that inhibition of CaMKII activity results in an upregulation of CaMKIV mRNA and protein in leukemia cell lines. Conversely, expression of CaMKIV inhibits autophosphorylation and activation of CaMKII, and elicits G0/G1cell cycle arrest,impairing cell proliferation. Furthermore, U937 cells expressing CaMKIV show elevated levels of Cdk inhibitors p27(kip1) and p16(ink4a) and reduced levels of cyclins A, B1 and D1. These findings were also confirmed in the K562 leukemic cell line. The relationship between CaMKII and CaMKIV is also observed in primary acute myeloid leukemia (AML) cells, and it correlates with their immunophenotypic profile. Indeed, immature MO/M1 AML showed increased CaMKIV expression and decreased pCaMKII, whereas highly differentiated M4/M5 AML showed decreased CaMKIV expression and increased pCaMKII levels. Our data reveal a novel cross-talk between CaMKII and CaMKIV and suggest that CaMKII suppresses the expression of CaMKIV to promote leukemia cell proliferation.