Laura M. Calvi - US grants

DESCRIPTION (provided by the applicant): Parathyroid hormone (PTH) has a complex and only partially understood action on bone, which mainly targets the osteoblast lineage. Many of the actions of PTH on bone are mediated by the PTH/PTHrP receptor (PPR). The proposed study aims to investigate the regulation of osteoblasts commitment, proliferation, differentiation and death by the PPR, as well as regulation of osteoclastogenesis and hematopoiesis by cells at different stages of maturation in the osteoblast lineage. In order to study these questions, cells from transgenic mice in which a constitutively active form of the PPR is expressed in the osteoblast lineage will be isolated and characterized. These mutant mice have a striking phenotype, with enormous increases in bone, osteoblasts, stromal cells and osteoclasts. This project will use an in vitro system to understand what these cells are shown to do in vivo. By studying the behavior of these cells, this project aims to define the cellular and molecular mechanisms that mediate the role of the PPR in osteoblasts. In addition, an improved understanding of the effects of PTH may provide the basis for advances in the treatment of common forms of metabolic bone disease, such as osteoporosis. The candidate states that the research project and the didactic activities proposed will help her acquire the expertise necessary to pursue a career focused on laboratory-based research of clinical relevance. Through analysis of the data obtained, discussion with her mentors and formal didactic training, she will be able to expand her knowledge of molecular genetics and development and become experienced in the field of cell biology, cell-cell interaction and signaling. Support from a KO8 award will assist in the completion of thi s proposal, and help the applicant to set out independently as an investigator, clinician and teacher in the field of PTH action and bone and mineral metabolism.

DESCRIPTION (provided by applicant): The bone marrow stromal microenvironment is a complex cellular network essential for the control of hematopoietic stem cells (HSC), primitive cells giving rise to all blood cell lineages throughout the life of an individual. In the bone marrow, HSC are found in close proximity to osteoblasts, bone-forming cells which are the main target of parathyroid hormone (PTH) in bone, through activation of the PTH/PTHrP receptor (PPR). Definition of HSC niche cellular components and of the mechanisms by which they influence HSC behavior has been difficult. Recent studies have shown that osteoblastic cells play a crucial role in HSC regulation and that osteoblastic PPR activation expands HSC. We have used PTH-treated mice as well as transgenic mice in which a constitutively active PPR is targeted to osteoblastic cells (col1-caPPR) to show that activation of the PPR in osteoblastic cells through Jagged1-Notch signaling doubles hematopoietic stem cell numbers. The primary goal of this proposal is therefore to determine the function of osteoblastic Jagged1 in PPR-mediated HSC expansion. We hypothesize that osteoblastic Jagged1 expression is necessary for PPR-depended HSC expansion, and that it is instrumental in expanding a specific subpopulation of osteoblastic cells, which best support HSC. These hypotheses will be examined through experiments designed to: 1) delineate PPR-dependent stromal osteoblastic Jagged1 expression; 2) determine whether PPR-mediated HSC expansion is controlled by osteoblastic Jagged1; and 3) define the effects of Notch signaling on the HSC microenvironment. These studies will determine the role of PTH-dependent Jagged1 and Notch signaling in the HSC niche, advance the understanding the basic mechanisms underlying the bone marrow microenvironmental control of HSC self-renewal, and provide the basis for developing novel approaches to facilitate HSC expansion, with the goal of improving clinical recovery from bone marrow transplantation and bone marrow failure states.

[unreadable] DESCRIPTION (provided by applicant): The microenvironment, or niche, in which hematopoietic stem cells (HSC) reside, is essential for their regulation. Since HSC number limits their clinical use, strategies to increase HSC through niche manipulation could increase the scope of their therapeutic application. The long-term objective of this proposal is to manipulate the bone marrow microenvironment to increase HSC numbers and thereby their clinical utility. To meet this objective, we established a novel experimental model in which treatment with parathyroid hormone (PTH) and/or activation of its receptor (PTH1R) in osteoblastic cells expands Long-term HSC (LT-HSC), thus identifying osteoblastic cells as key regulators of HSC. While defining mediators of PTH action in the niche, we discovered that Prostaglandin E2 (PGE2), which is released by osteoblastic cells upon PTH treatment, selectively expands Short-term HSC (ST-HSC) in vivo. Such exquisite and specialized regulation of HSC subsets has not previously been achieved, but we believe that it will have important therapeutic implications. Therefore, we have developed two unique experimental tools to define HSC regulation. Based on our observations, we hypothesize that independent mechanisms selectively regulate LT-HSC and ST-HSC and that these have different effects on HSC regulation. To demonstrate this hypothesis, this proposal will pursue three specific aims. In Aim1, the specific cellular and molecular mechanisms that mediate LT-HSC expansion in response to PTH will be defined in vivo by pharmacologic and genetic means. In Aim2, the specific cellular and molecular mechanisms that mediate PGE2-dependent ST-HSC expansion will be defined in vivo and in vitro. In Aim 3, interactions between regulation of LT-HSC and ST-HSC will be established. Data from the studies proposed in this application will 1) define the PTH and PGE2-activated mechanisms regulating LT-HSC and ST-HSC; 2) identify interactions between these mechanisms which could be exploited for targeted regulation of HSC subsets in the setting of specific therapeutic need. The studies proposed are therefore designed to result in findings that not only advance the understanding of stem cell regulation, but also devise pharmacologic strategies that can be brought back to patients, directly impacting their clinical care. The availability of PTH and PGE2 receptor specific agonists makes these studies particularly timely for future translation to human therapy. Using two novel in vivo experimental tools, this project will determine specific regulatory mechanisms, direct and indirect, that control HSC behavior, and that can stimulate differentially subsets of HSC which have different properties. Since HSC give rise to all blood cells, these regulatory mechanisms could be therapeutically exploited to increase HSC in specific situations of blood cell injury or deficiency. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to define the cellular and molecular mediators crucial for osteoblastic control of HSCs. We previously established that parathyroid hormone (PTH) activates osteoblastic cells to increase hematopoietic stem cell (HSC) numbers and that PTH improves HSC survival after radiation injury. These results give us a model to define a novel therapeutic approach to increase HSCs following iatrogenic or toxic injury to the bone marrow by stimulating osteoblastic cells. However, the specific osteoblastic cell subsets and the key osteoblastic-dependent molecular events regulating HSCs are unknown. Using pharmacologic and genetic models, we have identified Notch signaling as a potential mediator of PTH-dependent HSC regulation. Notch activation requires direct interaction of cell-bound ligands with receptors on neighboring cells. We demonstrated that 1) PTH or activation of its receptor stimulate the Notch ligand Jagged1 (Jag1) in osteoblastic cells; 2) in mice with constitutively active PTH receptors in osteoblastic cells, HSCs have increased Notch activation; 3) the PTH-dependent HSC increase is blocked by inhibition of ?-secretase activity, which is required for Notch activation. Our preliminary studies now demonstrate that expression of Jag1 in osteoblastic cells is required for the PTH-dependent HSC expansion. Together, these data suggest that PTH expands HSC through osteoblastic expression of Jag1, which then activates Notch signaling in neighboring bone marrow cells. Based on our data, we hypothesize that HSC expansion by osteoblasts requires Jag1-initiated Notch activation in the bone marrow microenvironment. To test this hypothesis, in Aim1 we will define the osteoblastic cell subset in which Jag1 is necessary and sufficient to mediate HSC expansion. In Aim2, we will identify the cell population (HSC, osteoblastic cells and/or other components of the bone marrow) in which Notch activation is required to achieve osteoblastic-dependent HSC expansion. Finally in Aim3 we will determine the contribution of Notch signaling to the myeloprotective effects of PTH, a clinical scenario in which HSC niche manipulation could be a novel strategy to reduce morbidity and mortality. We have already established and fully characterized in vivo models in which microenvironmental signals increase HSCs. Now that osteoblastic Jag1 has been identified as a key element of PTH-dependent HSC expansion, we have the unprecedented opportunity of defining the cellular and molecular components of the HSC niche using the in vivo strategies proposed here. Completion of our experimental aims will thus define novel therapeutic targets for HSC manipulation in the bone marrow microenvironment, which can be exploited to improve survival after bone marrow injury. PUBLIC HEALTH RELEVANCE: In this proposal, we study the regulation of hematopoietic stem cells (HSC) by their bone marrow microenvironment. Since HSC give rise to all blood cells, these regulatory mechanisms could be therapeutically exploited to increase HSC in specific situations of blood cell injury or deficiency.

DESCRIPTION (provided by applicant): Aging of the hematopoietic system results in an increased risk of developing cytopenias, Myelodysplastic syndromes (MDS), myeloproliferative disorders and leukemias. The Calvi laboratory and others have demonstrated the central role of osteoblastic lineage cells, a critical component of the marrow microenvironment, in normal hematopoietic stem and progenitor cell (HSPC) regulation. Aging changes HSPCs in both murine models and humans, decreasing their quiescence and their ability to reconstitute the marrow, and is thought to contribute importantly to aging of the whole hematopoietic system. Data are beginning to emerge supporting a role of the aging microenvironment on HSPC aging. However, this has not been described in humans, and it is unknown whether stimulation of the niche will reverse or mitigate the effects of aging. We have recently demonstrated that 2 pharmacologic agents which stimulate the marrow microenvironment (Parathyroid hormone and Prostaglandin E2) rapidly increase HSPC quiescence and stimulate the HSC niche in vivo. Based on these results, we hypothesize that aging of the niche contributes to HSPC dysfunction and that these changes can be remediated by niche stimulation. In this application, we propose a highly integrated and translational approach in which we will use young and aged human samples and murine experiments as well as 2 murine models of MDS to verify our central hypothesis and determine if use of treatments previously demonstrated to increase HSCs through microenvironmental stimulation can remediate age-dependent changes in the niche and /or in HSPCs. In this project we will determine if 1) HSPCs become dysfunctional in the setting of aging as a result of the aging niche and 2) targeted modulation of niche cells in the context of aging can improve HSPC support and prevent progression to MDS-related bone marrow failure. Completion of these aims will elucidate a novel therapeutic strategy to reverse marrow failure and susceptibility to leukemia in older adults.

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop a novel therapy - prostaglandin E2 (PGE2) - to mitigate the effects of radiation on the hematopoietic system. This robust cellular system is disrupted and damaged by exposure to radiation, which results in cytopenias leading to life-threatening infections, anemia, and bleeding. The onset and extent of platelet loss predicts survival following total body irradiation. The majority of agents that have been developed and stockpiled for use as part of a radiological emergency are specifically targeted at the clinical consequences of white blood cell loss. Although much work has been done over the past decades, few agents have transitioned from bench to clinic. The collaborative studies of the Palis and Calvi labs within the University of Rochester's Center for Medical Countermeasures against Radiation have resulted in the development of robust mouse models of acute and late radiation injury. Our preliminary studies indicate that PGE2, delivered 48-72 hours after acute radiation exposure, mitigates the megakaryocyte lineage leading to more rapid platelet recovery. In addition, PGE2 acutely mitigates the number of phenotypic HSC. We hypothesize that PGE2 acts through the marrow microenvironment, specifically at the level of endothelial cells and macrophage populations, to mitigate radiation-induced injury of hematopoietic stem cells and megakaryocyte precursors. We will assess the effectiveness and mechanism of PGE2 mitigation of acute and late hematologic injury. Our joint studies have also identified a special population - 14 day old mice - as being particularly sensitive to relatively lw sublethal radiation exposure, since they develop not only a severe reduction in phenotypic HSCs but also late peripheral cytopenias. A better understanding of the differential response between pediatric and adult populations, both to radiation injury and any proposed agents, will be required to develop treatments with broad applicability. We will, therefore, also investigate the efficacy of PGE2 to mitigate late injury to the hematopoietic system of pediatric versus adult populations. Taken together, our proposed collaborative, mechanistic studies will bring forward PGE2, a promising new agent with an established safety profile, for use in mitigating both acute and late effects of radiation exposure.

DESCRIPTION (provided by applicant): The mechanisms by which a leukemic clone suppresses normal hematopoiesis are poorly understood, and yet this phenomenon likely contributes to disease progression, disease morbidity and response to therapy. Our recent analysis of the bone marrow microenvironment (BME) in a syngeneic mouse model of acute myeloid leukemia1 demonstrated dramatic osteoblastic defects. Since our laboratory and others have demonstrated the central role of osteoblastic lineage cells in hematopoietic stem cell (HSC) regulation, these data identify osteoblastic cells as a potential clinical target to stimulat normal HSC recovery in leukemia and decrease BME support of leukemic stem cells (LSCs). Moreover, we discovered leukemic production of the chemokine CCL3, recently demonstrated to inhibit osteoblastic function in multiple myeloma. With the long-term goal of targeting the HSC and leukemia stem cell (LSC) niches to improve therapy for leukemia and impact disease control, the current proposal aims to efficiently, effectively and safely apply pharmacologic tools currently approved for bone anabolic treatment to leukemia. We hypothesize that 1) leukemia cells decrease the ME support of HSCs and normal hematopoiesis in favor of LSCs and bulk leukemia, promoting disease progression and that 2) interference with leukemia signals disrupting the ME and/or ME stimulation by bone anabolic treatment in the context of leukemia will improve HSC support and decrease LSC competitiveness. Using two murine models as well as a novel method of isolation of osteoblastic cells from spicules in normal and leukemic human bone marrow samples, we propose to: 1.) Define the extent and timing of leukemia-induced osteoblastic lineage inhibition. 2.) Define changes on leukemia-induced ME ability to support normal and malignant hematopoiesis. 3.) Establish the requirement for CCL3 as the mediator of leukemia-induced ME changes using loss of function, overexpression and pharmacologic approaches. 4.) Determine if therapeutic targeting of leukemia-associated osteoblasts impacts normal hematopoiesis, disease progression and LSC function. Data from this project would represent a paradigm shift in the therapy for patients with AML, where targeting of the BME improves our ability to treat the leukemia and more readily restore normal hematopoiesis. Agents stimulating bone forming cells are already available for patient use in the non-malignant scenario allowing for rapid translation into the clinic.

The program entitled ?Rochester Musculoskeletal (ROCMSK) Training Program? at the University of Rochester Medical Center is designed to providing interdisciplinary didactic and research training in musculoskeletal science. The overarching aim of ROCMSK Training Program is to develop future generations of interdisciplinary musculoskeletal scientists and leaders of innovations. The program will be administered in The Center for Musculoskeletal Research (CMSR) at the University of Rochester and integrates 21 highly- collaborative faculty with primary appointments in 7 academic and clinical departments. ROCMSK proposes slots for 4 predoctoral and 2 postdoctoral trainees. The education program detailed herein ensures a comprehensive understanding of musculoskeletal science that is seamlessly accessible to all CMSR trainees at every academic level. The training experience aims to build competency in areas ranging from the most basic molecular and genetic studies to the design and execution of human clinical trials. The CMSR and associated training faculty represent a highly integrated group of Mentors that provide research training opportunities in Bone Biology and Disease, Cartilage Mechanobiology, Arthritis, and Regenerative Therapies, Tendon Development, Repair and Regenerative Engineering, Muscle Biology and Disease, Drug Delivery, Fracture Repair and Bone Tissue Engineering, Musculoskeletal Infection, Stem Cells and Musculoskeletal Development and Regenerative Biology, and Skeletal Cancer Biology and Therapeutics. Accepted predoctoral trainees matriculate into one of 6 degree-awarding departments and programs at the University of Rochester. After matching in a ROCMSK mentor lab, Training grant eligible (TGE) students can apply for a T32 award and, if selected, can join the Program. TGE Postdocs will be recruited by ROCMSK mentors, after which they are immediately T32 training seat eligible. A hierarchy of oversight committees led by the MPI team are organized to efficiently manage all training activities. ROCMSK training will emphasize basic and translational science education through: contemporary curricular activities specifically designed for musculoskeletal science; innovative research experiences pursued through PhD dissertations and post-doctoral projects with emphasis on scientific rigor and biomedical ethics; individualized development plans that utilize evidence-based approaches for career planning; expansive networking opportunities through monthly invited-speaker seminars and an annual symposium; and scientific communication through formal expectations of publications and presentations at national meetings. These activities, which are ongoing within the Center but will be enhanced through ROCMSK, will attract outstanding young scientists to the program. Overall, the training enabled through ROCMSK, which includes a focus on mentoring trainees towards independent funding, will be a springboard to development into highly successful, collaborative musculoskeletal investigators empowered to translate basic discoveries into human therapies.