Hiranmoy Das - US grants

[unreadable] DESCRIPTION (provided by applicant): This proposal describes a 5 year training program for the development of an academic career in Cardiovascular Transcriptional Biology. The principal investigator has scientific background with expertise in cellular immunology. He will now embark upon a program which is particularly designed to expand his scientific experience, such that he develops the skills required for an independent research career. This program will provide in-depth knowledge and experience in molecular biology, in particular transcriptional regulation, and in transgenic animal studies. Dr. Mukesh K. Jain, MD, will mentor the principal investigator's scientific development. Dr. Jain has a very active laboratory and is the Director of Cardiovascular Research Institute, Case Western Reserve University, Cleveland OH. He has trained several research fellows. The candidate's training will be enhanced by the collaboration with Prof. Diane Mathis, PhD - an expert in the field of arthritis and inflammation research. The project is designed to determine the effect of the transcription factor KLF2 on monocyte activation and function. Preliminary data demonstrate that this factor is expressed in monocytes, and reduced upon cytokine activation or differentiation. Furthermore, forced overexpression of KLF2 in monocytes inhibits the LPS-mediated induction of proinflammatory factors, cytokines and chemokines and reduced phagocytosis. Conversely, short interfering RNA-mediated reduction in KLF2 increased inflammatory gene expression. Mechanistically, KLF2 inhibits the transcriptional activity of both NF-kB and activator protein-1 in part by means of recruitment of transcriptional coactivator p300/CBP-associated factor, PCAF. These observations implicate KLF2 as a novel negative regulator of monocytic activation. The proposed experiments in this application will build on these initial observations and explore them in depth - both mechanistically and in models of arthritis in vivo. Studies will include a detailed analysis of effect on target genes with KLF2 overexpression (by adenoviral overexpression and transgenic models) and siRNA-mediated 'knockdown' approaches. KLF2 transgenic mice will be studied to assess the effects of this factor on the development of arthritis in vivo. Proposal was made to generate acute/ chronic arthritis in KLF2 transgenic mice and will be determined their biological consequences by evaluating arthritis development, macrophage recruitment, immunohistochemistry and gene expression during arthritis development. The monocyte is a key cell type in the regulation of the body's inflammatory response. The applicant's identification of the factor KLF2 as a key regulator of monocyte activation may provide the basis for novel therapies directed at limiting inflammation for a broad spectrum of disease states including arthritis. [unreadable] [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): The long-term objective of this proposal is to investigate the role of zinc finger transcriptional factor, Kruppel like factor-2 (KLF2) in the regulation of osteoclastogenesis and rheumatoid arthritis (RA)-related pathogenesis. Among the cellular components monocytes play major roles in mediating progression of RA. The monocytes migrate from the peripheral blood to the arthritic joint tissues and secrete pro-inflammatory factors, and these factors in turn mediate inflammation and recruit other immune cells, which mediate differentiation of monocytes towards osteoclasts. Since it has been reported that KLF2 can induce quiescence to the immune cells and also inactivate monocytes in response to stimulation (active monocytes are required to promote osteoclastogenesis) and because our preliminary results indicate severe arthritic joint changes in KLF2 hemizygous mice (KLF2 homozygous knock out mice are embryonically lethal), we therefore hypothesize that this transcription factor may inhibit osteoclastogenesis and thereby, pathogenesis of RA. Aim1 will determine the importance of myeloid KLF2 in regulating the pathogenesis in RA. Experimental Design: Conditional gain and loss of gene function approach will be undertaken to determine the role of KLF2 in the regulation of pathogenesis in vivo. Complementary histologic, histomorphometric, immunohistochemical, and radiologic (micro CT) studies will be used to evaluate extent of the disease. Human monocytes and joint tissues from RA and healthy controls will be studied to confirm findings. Aim 2 will examine the bases for KLF2's ability to regulate osteoclastogenesis. Experimental Design: Gain- and loss-of-function approaches will be used to determine the role of KLF2 in osteoclastic differentiation. Additionally, the cross talk between KLF2 and osteoclastogenesis markers (NFATc1, NF?B, Cathepsin K and MMP9) and osteoblastogenesis markers (RANK/RANKL, OPG, BMP2/4, and Runx2) will be determined. Human monocytes from RA and healthy controls will also be studied to confirm findings. Aim 3 will investigate whether the effects of pharmacological compound on arthritis and osteoclastogenesis are KLF2 dependent. We found that a group of pharmacological compound, such as HDAC inhibitor (HDACi) induces KLF2 expression in myeloid cells. Experimental Design: We will verify if HDACi induce KLF2 in myeloid cells in mice. Next, we will determine if the HDACi-mediated regulation of inflammation and osteoclastogenesis is KLF2- dependent using various molecular approaches (structure-functions studies, co-immunoprecipitation, and ChIP assays). The knowledge generated from this study will not only identify a novel endogenous regulator of osteoclastogenesis in RA, but also indicate newer and effective strategies to control this disease.

Summary Glucocorticoid-induced osteoporosis is the most common cause of secondary osteoporosis, and the primary cause before age 50. It affects over 0.5% of the general population, wherein 65% are women. In post- menopausal women and men aged 50 and over, treatment would be recommended in at least half based on the 2010 American College of Rheumatology guidelines. Adult stem cells are a promising class of regenerative cells, some lineages of which can be induced to differentiate into osteoblasts and potentially cure osteoporosis. We have shown that 250-fold nanofiber-expanded hematopoietic (CD34+) stem cells can reverse glucocorticoid-induced osteoporosis in an aged murine model. Now we have discovered a way to expand CD34+ stem cells over a million-fold while preserving their stemness, which promises to make this therapy cost effective. These cells can be obtained noninvasively from umbilical cord blood (UCB). UCB is readily available and is considered safe for human application for treating a variety of diseases such as leukemia and other blood disorders. If these million-fold expanded CD34+ stem cells can be shown to be safe and efficacious in reverting glucocorticoid-induced osteoporosis, this cell-based therapy may extend to other types of osteoporosis, and would represent a major paradigm shift. Patients with osteoporosis must undergo lifetime therapy with traditional therapeutics, whereas stem cells promise to be a 1 to 2 time treatment. Furthermore, patients will not have extended hospitalization nor surgery costs, as stem cell therapy in many cases is an outpatient IV infusion. Million-fold expansion provides the opportunity to reduce costs further, as one cord blood unit will be able to treat over 100 patients. The aims of this Phase I project are therefore to (a) determine the potential of million-fold expanded CD34+ stem cells to differentiate into osteoblasts, and (b) test the efficacy of million-fold expanded CD34+ stem cells for reverting glucocorticoid-induced osteoporosis in aged mice. If successful, this research will be extended to more severe forms of osteoporosis, and potentially provide a pathway towards a cost effective clinical regenerative treatment for this debilitating disease. It may also have extensions for the lower-cost treatment of leukemias and a variety of other blood disorders, which will be the subject of future investigations.