Ewing sarcoma (ES) involves a tumor-specific chromosomal translocation that produces the EWS-FLI1 protein, which is required for the growth of ES cells both in vitro and in vivo. However, an EWS-FLI1-driven transgenic mouse model is not currently available. Here, we present data from six independent laboratories seeking an alternative approach to express EWS-FLI1 in different murine tissues. We used the Runx2, Col1a2.3, Col1a3.6, Prx1, CAG, Nse, NEFL, Dermo1, P0, Sox9 and Osterix promoters to target EWS-FLI1 or Cre expression. Additional approaches included the induction of an endogenous chromosomal translocation, in utero knock-in, and the injection of Cre-expressing adenovirus to induce EWS-FLI1 expression locally in multiple lineages. Most models resulted in embryonic lethality or developmental defects. EWS-FLI1-induced apoptosis, promoter leakiness, the lack of potential cofactors, and the difficulty of expressing EWS-FLI1 in specific sites were considered the primary reasons for the failed attempts to create a transgenic mouse model of ES.

INTRODUCTION:Insulin like growth factor (IGF)-I can act on a variety of cells involved in cartilage and bone repair, yet IGF-I has not been studied extensively in the context of inflammatory arthritis. The objective of this study was to investigate whether IGF-I overexpression in the osteoblast lineage could lead to increased reparative or pathological bone formation in rheumatoid arthritis and/or spondyloarthritis respectively.METHODS:Mice overexpressing IGF-I in the osteoblast lineage (Ob-IGF-I+/-) line 324-7 were studied during collagen induced arthritis and in the DBA/1 aging model for ankylosing enthesitis. Mice were scored clinically and peripheral joints were analysed histologically for the presence of hypertrophic chondrocytes and osteocalcin positive osteoblasts.RESULTS:90-100% of the mice developed CIA with no differences between the Ob-IGF-I+/- and non-transgenic littermates. Histological analysis revealed similar levels of hypertrophic chondrocytes and osteocalcin positive osteoblasts in the ankle joints. In the DBA/1 aging model for ankylosing enthesitis 60% of the mice in both groups had a clinical score 1<. Severity was similar between both groups. Histological analysis revealed the presence of hypertrophic chondrocytes and osteocalcin positive osteoblasts in the toes in equal levels.CONCLUSION:Overexpression of IGF-I in the osteoblast lineage does not contribute to an increase in repair of erosions or syndesmophyte formation in mouse models for destructive and remodeling arthritis.

Peroxisome proliferator-activated receptor gamma (PPAR gamma), known as the master regulator of adipogenesis, has been regarded as a promising target for new anti-osteoporosis therapy due to its role in regulating bone marrow mesenchymal stem/progenitor cell (BMSC) lineage commitment. However, the precise mechanism underlying PPAR gamma regulation of bone is not clear as a bone-specific PPAR gamma conditional knockout (cKO) study has not been conducted and evidence showed that deletion of PPAR gamma in other tissues also have profound effect on bone. In this study, we show that mice deficiency of PPAR gamma in cells expressing a 3.6 kb type I collagen promoter fragment (PPAR(fl/fl):Col3.6-Cre) exhibits a moderate, site-dependent bone mass phenotype. In vitro studies showed that adipogenesis is abolished completely and osteoblastogenesis increased significantly in both primary bone marrow culture and the BMSCs isolated from PPAR gamma cKO mice. Histology and histomorphometry studies revealed significant increases in the numbers of osteoblasts and surface in the PPAR gamma cKO mice. Finally, we found that neither the differentiation nor the function of osteoclasts was affected in the PPAR gamma cKO mice. Together, our studies indicate that PPAR gamma plays an important role in bone remodeling by increasing the abundance of osteoblasts for repair, but not during skeletal development. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

We generated a new Bmp2 conditional knock-out allele without a neo cassette and removed Bmp2 gene in osteoblasts (Bmp2-cKOob) using the 3.6Col1a1-Cre transgenic model. Bones of Bmp2-cKOob mice are thinner, with increased brittleness. Osteoblast activity is reduced as reflected in reduced bone formation rate, and failure to differentiate to a mature mineralizing stage. Bmp2 in osteoblasts also indirectly controls angiogenesis in the periosteum and bone marrow. VegfA production is reduced in Bmp2-cKOob osteoblasts. Deletion of Bmp2 in osteoblasts also leads to defective mesenchymal stem cells (MSC), which correlates with the reduced microvascular bed in the periosteum and trabecular bones. Several marker genes of MSC (α-SMA, CD146 and Angiopoietin-1), in vitro CFU assays and deletion of the Bmp2 gene in vitro in α-SMA+ BMSC support our conclusions. Critical roles of the Bmp2 gene in osteoblasts and MSC are a vital link between bone formation, vascularization and mesenchymal stem cells.

Glycogen synthase kinase 3 β (GSK-3β) is an essential negative regulator or "brake" on many anabolic-signaling pathways including Wnt and insulin. Global deletion of GSK-3β results in perinatal lethality and various skeletal defects. The goal of our research was to determine GSK-3β cell-autonomous effects and postnatal roles in the skeleton. We used the 3.6-kb Col1a1 promoter to inactivate the Gsk3b gene (Col1a1-Gsk3b knockout) in skeletal cells. Mutant mice exhibit decreased body fat and postnatal bone growth, as well as delayed development of several skeletal elements. Surprisingly, the mutant mice display decreased circulating glucose and insulin levels despite normal expression of GSK-3β in metabolic tissues. We showed that these effects are due to an increase in global insulin sensitivity. Most of the male mutant mice died after weaning. Prior to death, blood glucose changed from low to high, suggesting a possible switch from insulin sensitivity to resistance. These male mice die with extremely large bladders that are preceded by damage to the urogenital tract, defects that are also seen type 2 diabetes. Our data suggest that skeletal-specific deletion of GSK-3β affects global metabolism and sensitizes male mice to developing type 2 diabetes.

cAMP signalling is both a major pathway as well as a key therapeutic target for inducing immune tolerance and is involved in Treg cell (regulatory T-cell) function. To achieve potent immunoregulation, cAMP can act through several downstream effectors. One proposed mechanism is that cAMP-mediated suppression, including immunosuppression by Treg cells, results from activation of PKA (protein kinase A) leading to the induction of the transcription factor ICER (inducible cAMP early repressor). In the present study, we examined CD4(+)CD25(-) Teff cell (effector T-cell) and CD4(+)CD25(+) Treg cell immune responses in Crem (cAMP-response-element modulator) gene-deficient mice which lack ICER (Crem(-/-)/ICER-deficient mice). ICER deficiency did not significantly alter the frequency or number of Treg cells and Teff cells. Treg cells or a pharmacological increase in cAMP suppressed Teff cells from Crem(+/+) and Crem(-/-)/ICER-deficient mice to an equivalent degree, demonstrating that ICER is dispensable in these functions. Additionally, activating the cAMP effector Epac (exchange protein directly activated by cAMP) suppressed Teff cells. Treg cells expressed low levels of all cyclic nucleotide Pde (phosphodiesterase) genes tested, but high levels of Epac. These data identify ICER as a redundant mediator of Treg cells and cAMP action on Teff cells and suggest that Epac may function as an alternative effector to promote cAMP-dependent Teff cell suppression.

While the epidermal growth factor receptor (EGFR)-mediated signaling pathway has been shown to have vital roles in many developmental and pathologic processes, its functions in the development and homeostasis of the skeletal system has been poorly defined. To address its in vivo role, we constructed transgenic and pharmacologic mouse models and used peripheral quantitative computed tomography (pQCT), micro-computed tomography (µCT) and histomorphometry to analyze their trabecular and cortical bone phenotypes. We initially deleted the EGFR in preosteoblasts/osteoblasts using a Cre/loxP system (Col-Cre Egfr(f/f)), but no bone phenotype was observed because of incomplete deletion of the Egfr genomic locus. To further reduce the remaining osteoblastic EGFR activity, we introduced an EGFR dominant-negative allele, Wa5, and generated Col-Cre Egfr(Wa5/f) mice. At 3 and 7 months of age, both male and female mice exhibited a remarkable decrease in tibial trabecular bone mass with abnormalities in trabecular number and thickness. Histologic analyses revealed decreases in osteoblast number and mineralization activity and an increase in osteoclast number. Significant increases in trabecular pattern factor and structural model index indicate that trabecular microarchitecture was altered. The femurs of these mice were shorter and smaller with reduced cortical area and periosteal perimeter. Moreover, colony-forming unit-fibroblast (CFU-F) assay indicates that these mice had fewer bone marrow mesenchymal stem cells and committed progenitors. Similarly, administration of an EGFR inhibitor into wild-type mice caused a significant reduction in trabecular bone volume. In contrast, Egfr(Dsk5/+) mice with a constitutively active EGFR allele displayed increases in trabecular and cortical bone content. Taken together, these data demonstrate that the EGFR signaling pathway is an important bone regulator and that it primarily plays an anabolic role in bone metabolism.

The expression of inducible cAMP early repressor (ICER) has been demonstrated in cultured osteoblastic MC3T3-E1 cells, calvarial cultures and in vivo, mainly via the cAMP-PKA signaling pathway. To determine the molecular mechanism(s) for this action, a fragment of the CREM P2 promoter from -238 bp to +14 bp was linked to a luciferase reporter (CREMP2-Luc238). This region contains 2 clusters of cAMP-responsive element (CRE). The 5’ cluster contains CRE1 and CRE2 while the 3’ cluster contains CRE3 and CRE4 and the two clusters are separated by 12 bp. In order to study if any or all CREs are responsive to cAMP induction, osteoblastic MC3T3-E1 cells were stably transfected with constructs having serial deletions of each CRE or mutations in each cluster of the CREMP2-Luc construct. Agonists that stimulate the cAMP-PKA pathway induced CREMP2-Luc238 activity, and neither PKC nor Ca++ pathways did. The deletion of CRE1 and CRE2 did not significantly abolish the cAMP inducibility of CREMP2-Luc238 activity. Electrophoretic mobility shift assay showed many DNA/protein complexes when probes from either CRE1-2 or CRE3-4 were incubated with FSK-treated nuclear extract from MC3T3-E1 cells. However, only a small portion of the complexes was competed by a consensus CRE oligonucleotide. Interestingly, a major DNA/protein complex seen in the binding of CRE3-4 was not competed by the consensus CRE oligonucleotide, but by a mutated CRE oligonucleotide suggesting that other transcription factor(s) may be involved in the binding of CRE3-4. The CRE binding DNA/protein complexes were supershifted mainly by antibodies against ICER and CREB, mildly by C/EBPβ suggesting these transcription factors may be involved in the FSK induction of the P2 promoter of the CREM gene.

Protein deficiency is frequently observed in elderly osteoporotic patients. Undernutrition leads to decreased levels of IGF-I, an important factor in regulating bone homeostasis throughout life. IGF-I is produced in the liver and locally in the skeleton. We hypothesized that increasing IGF-I expression in the osteoblasts, the bone forming cells, would protect the skeleton from the negative effects of a low-protein diet. To test our hypothesis, we employed a mouse model in which IGF-I was overexpressed exclusively in osteoblasts and fed either a 15% (normal) or a 2.5% (low) protein isocaloric diet to the transgenic (TG) mice and their wild-type (WT) littermates for 8 weeks. Blood was collected for biochemical determinations and weight was monitored weekly. Bones were excised for microstructural analysis (μCT), as well as biomechanical and material level properties. Histomorphometric analysis was performed for bone formation parameters. A low protein diet decreased body weight, circulating IGF-I and osteocalcin levels regardless of genotype. Overexpression of IGF-I in the osteoblasts was, however, able to protect the negative effects of low protein diet on microstructure including tibia cortical thickness and volumetric density, and on bone strength. Overexpression of IGF-I in osteoblasts in these mice protected the vertebrae from the substantial negative effects of low protein on the material level properties as measured my nanoindentation. TG mice also had larger overall geometric properties than WT mice regardless of diet. This study provides evidence that while a low protein diet leads to decreased circulating IGF-I, altered microstructure and decreased bone strength, these negative effects can be prevented with IGF-I overexpression exclusively in bone cells.

No one person in the world of bone has had a greater impact than Larry on us all. To honor his memory, we asked some of Larry's closest friends and colleagues to reflect upon his extraordinary life. What follows is an expression of that meaning sprinkled with Larry's inimitable wit and reflections on life, science, and the future. “It is often said that something may survive of a person after his death, if that person was an artist and put a little of himself into his work. It is perhaps the same way that a sort of cutting taken from one person and grafted on to the heart of another continues to carry on its existence even when the person from whom it had been detached has perished.” Proust, Remembrance of Things Past Larry was born in New York City where his father was a doctoral student in geology at Columbia University and his mother was a student at Columbia's Teachers College. He grew up in a family environment that emphasized intellectual achievement and was presented with wonderful opportunities for his formal education at outstanding institutions in Cambridge and Boston, Massachusetts. At Browne and Nichols School, he was “intensively prepared for entering Harvard College.” Larry wrote a vignette of that preparatory school experience: “My English teacher, Jimmy Reeves, despaired at my inability to tell the difference between a comma and a semicolon. He told me that every time my theme had a ‘comma fault’ he would reduce my grade by 5 points. When the grade reached zero, he gave up.” When Larry entered Harvard College in 1942, at the age of 16, World War II was raging and those who ran our country thought it necessary to compress the time spent in university to assure a steady supply of young intelligent manpower. When he was 18, Larry wrote the following note in The Harvard Crimson, the student newspaper, on July 1, 1943: “A year ago last week Harvard began its greatest transition year in history from a peacetime University, rich in liberal tradition, to a wartime training center, turning out skilled men quickly and efficiently.” Note Larry's good sense of prose style and his great first (the lead) sentence. It could have been predicted that Larry would eventually publish many fine papers and books and be the editor of a scientific journal (JBMR) that would profoundly influence a new field. Following the relatively short time at Harvard College, Larry entered Harvard Medical School, where he was introduced to disorders of the “bony skeleton” by Fuller Albright. After graduation in 1947, he served his internship at Boston City Hospital (BCH). Dr. William B. Castle, a charismatic physician and noted hematologist, was chief of a vibrant Harvard Service at the BCH, and Larry chose to undertake his first postdoctoral clinical training there. The opportunities to learn clinical medicine and to be aware of the emerging scientific basis of the diseases he encountered were extraordinary. Larry continued his clinical training at the Boston Veterans Administration (VA) Hospital where Dr. Maurice (Maurie) Strauss was chief of medicine. Maurie Strauss was a noted hematologist and nephrologist, a great physician and teacher who, as described in a 1974 New England Journal of Medicine paper (Papper, NEJM, 1974), “had enormous impact on people” and who “opened students' eyes, minds and hearts.” Larry had been a postdoc with Homer Smith in the Department of Physiology at New York University in the late 1940 s, and his work there on regulation of extracellular fluid volume and salt metabolism resulted in several of his earliest publications. He continued as a nephrologist and renal physiologist when he returned to Boston a few years later to become chief of the Renal Section at the Boston VA Hospital. It may be difficult for current medical graduates who are usually pressured to make their specialty choices early in medical school to comprehend how different it was 50–60 years ago, when one's teachers and role models could profoundly influence the development of careers after graduation. It is likely that Dr. Strauss and others had a major influence on the direction of Larry's early career just as Larry subsequently had a major influence on the careers of many of his students and colleagues. Larry continued his studies on controls of renal function when he left Boston for a position at SUNY Upstate Medical Center in Syracuse. In 1956, he published his first bone paper on pseudopseudohypoparathyroidism in the American Journal of Medicine. This was it! Within a few years Larry's research became all bone and bone-related, with renal physiology and renal pathophysiology just parts of his past. One doesn't know whether Larry's rapid transition from kidney to bone was because he literally “fell in love” with bone or whether he excused his new passion by saying humbly that there were too many nephrologists and he was getting out of a field that was “too crowded.” It was clear, however, that Larry did not take the advice of Maurie Strauss, who said to him, “you should stay away from calcium, it's a jungle.” We do know that had he continued as a nephrologist, Larry would have been the same gentle, persuasive giant that he was in our field for half a century. In 1974, a small band of devotees to the emerging field of bone and mineral (Larry, Lou Avioli, Norman Bell, Claude Arnaud, John Potts, Bill Peck, and Shirley Hohl) met at the Drake Hotel in Chicago to consider the idea that the field needed a new society to advance this newly recognized scientific discipline. The meeting marked the birth of the American Society for Bone and Mineral Research, followed by its incorporation in 1977 and its inaugural meeting at the Disneyland Hotel in Anaheim, California, in June 1979. Shirley Hohl was the first executive director. At that first meeting, 147 people came for one day to hear, in one room, a few plenary lectures and to discuss the posters that lined the back and sides of the room. The meeting was declared a success. And indeed it was, although objectively, one couldn't be so sure at the time. In fact, the new society had no money. It was Larry's mother who lent the ASBMR $4,000 to start it! Larry was the second president of the ASBMR (1980–1981). He was truly a leader extraordinaire, who led with his actions and whom others inevitably and irresistibly followed. One example of this leadership was the establishment of the JBMR. The question to which Larry clearly gave the answer was: When does a scientific society identify itself with a scientific journal? It was Larry, above all, who prevailed upon the ASBMR to see the wisdom of starting a journal within only a decade of the Society's founding. “Knowledge comes, but wisdom lingers.” Alfred, Lord Tennyson There was no question about who would be the first Editor-in-Chief of the JBMR. For a decade, Larry shepherded the journal on a path that quickly established it among the scientific elite in the bone field. Larry's standards were unwavering, but he also unashamedly encouraged, cajoled, and otherwise convinced biomedical scientists that they wanted to publish their best science in this journal. The editors of the JBMR will always stand in the shadow of Larry Raisz, whose scientific philosophy and personality set the Journal on a remarkable trajectory that continues to this day. On the Journal's 20th anniversary, Larry humbly remarked, “The journal succeeded because authors submitted good papers.” While this is undoubtedly true, in reality, investigators in bone and mineral felt honored, if not compelled, to submit to this new journal because Larry was its editor. Through the JBMR, he set an example of how to think about and conduct science. Larry conceived of a journal for the dissemination of bench-to-bedside science long before the words “translational research” entered our vocabulary. The inaugural issue covered topics ranging from the pathophysiology of osteoporosis to the emerging field of bone immunology. The juxtaposition of articles in clinical and basic science would remain a touchstone for the Journal and was conceived to reflect the diverse topics covered at the ASBMR annual meeting. All of this reflected Larry's devout belief that when clinical and basic science are presented together, new insights were more likely to arise. Larry's eclectic vision for publicizing bone and mineral science created a journal that embraced new ideas and approaches. In the first issue, he told readers he would seek out controversy. This message emboldened both junior and established investigators to pursue novel topics. Larry also stressed that the JBMR would be international in scope, an uncommon approach in the days before Internet communication. This openness led to the appointment of Associate Editors for the Journal who not only had broad-ranging and excellent scientific expertise, but also came from countries spanning the globe. Larry's vision and energy propelled the JBMR to the top rank of publications in the bone and mineral field. The direction he provided to “capture” in the pages of the Journal the fruits of the scientific revolution that began during his tenure set the course for the Journal's success. The content of the Journal continues to reflect Larry's original vision: cutting-edge physiology studies; investigations regarding new drug development; and studies resulting from the emergence of molecular biology, cell biology, genomics, and proteomics as established disciplines. Thus, the innovative nature of the Journal became a platform for its growth. It continues to echo the thrill and adventure he envisioned. No one understood bone and mineral biology as completely as Larry did. Larry read every paper before publication and every paper after publication. He often called one of us (JPB) after rereading the latest issue to tell him, as head of the Publications Committee, that this paper or that paper could have been written more clearly, could have been shorter, could have had the layout of its figures sized better, or simply to say, with a phone voice that exuded only joy, “What a great paper!” Is there any Editor-in-Chief in the world who can be said to be or to have been as focused, as diligent, and as utterly in love with what he was doing? Au Vieux Marc … We all knew the young Dr. Drezner With his rapier wit, he would take no prisoner. Those were the days when the boys from Duke Would win a game that wasn't a fluke. And Marc was another Durham Bull His life was rich, his plate was full. Alas he decided that he could edit A journal and we must give him credit. When an exhausted Larry handed it over Marc knew it would not be a bed of clover But he flourished right thought the transition And did great new things with each edition But also as JBMR's stature gained The Huskies waxed while the Blue Devils waned. As Marc builds the Journal and makes it great The glories of Durham seem to abate. Raisz, on the occasion of Marc Drezner replacing Larry as Editor-in-Chief of the JBMR As Larry's science merged more and more closely with osteoporosis, compelling opportunities arose to make this serious disease a household name. Larry became a founder and original board member of the National Osteoporosis Foundation in the United States. As a major advocacy group for osteoporosis, the NOF was fortunate to have Larry, who worked tirelessly to advance public awareness of this disease. The Surgeon General's report on bone health and osteoporosis in 2004 stands as a landmark to Larry, who was its scientific editor. The statement attributed to him, which formed the inspiration for this document, is “We know enough now to act on what we know.” This was typical of Larry. Always knowing more than anyone, he also knew when to stop, pause, and smell the roses of scientific advancement before plowing further the fertile grounds of discovery. Larry inspired the NOF with direction and a mission. The combination of his commitment to scientific excellence and patient care became a hallmark of the NOF as it drew strength from these principles. The NOF's successful outreach and educational resources can be attributed to Larry's leadership. After publication of the Surgeon General's report, Larry made an enormous effort to increase the recognition of a fracture as a “sentinel event.” Larry understood that there was an urgent public health requirement to improve the identification of the patient at high risk for fracture, and he worked to highlight the point that the postfracture patient was at the highest risk for subsequent fractures. As a part of the National Action Plan to bring the components of the Surgeon General's report to life, fracture as a sentinel event has become a major focus of attention. A meeting on health systems approaches for reducing the risk of new fractures in the post fracture patient was held in the fall of 2010 and was dedicated to Larry's memory. Those who joined him in this effort must now accept the responsibility of moving forward without his presence but with the history of his inspired leadership. Larry was the Board of Trustees Distinguished Professor of Medicine Emeritus at the University of Connecticut Health Center. He was instrumental in establishing the Lowell P. Weicker, Jr., General Clinical Research Center and served as its first program director from 1993 to 2002. He was director of the University of Connecticut Center for Osteoporosis, and in 2005 he became the first program director of the New England Musculoskeletal Institute at UConn. Larry's scientific contributions are discussed elsewhere, but it is important to remember Larry for his scientific leadership. He was the most visible attendee at the ASBMR Scientific Meeting, year after year after year, including his last meeting in the fall of 2009, when he was not well. Larry was the omnipresent questioner at the microphone after most presentations. Can one explain how Larry could pop up repeatedly, unashamedly, to ask a question that probably no one had thought of, while listening to the same presentation? If you sat next to him, you would know why. Larry had an uncanny ability to immerse himself immediately in the presentation; he was instantly in utter concentration listening to the abstract presentation, and when it was over, he was up, unable to control or contain himself, wanting to transmit to the author support, congratulations, and a great question. He would sit down, and immediately get ready for the next 10-minute jewel he was about to experience. His mind's eye had perfect vision. The microphone pulsed, time after time after time at ASBMR, in his inimitable voice: “Raisz, Connecticut.” Larry Raisz was a preeminent leader, investigator, mentor, teacher, and clinician in the field of bone and mineral metabolism for over half a century. Larry's research program on the mechanisms of bone formation and resorption and the pathogenesis of osteoporosis led to more than 450 original publications, reviews, and book chapters. Larry mentored over 100 students, postdoctoral trainees, clinical fellows, and faculty members. The interactions that formed these close associations were of paramount importance to him. At the SUNY Upstate Medical Center in Syracuse, his papers with Bill Au and others inaugurated the use of organ cultures to study the pathophysiology and biology of bone resorption. He continued this general approach with great success after he moved from Syracuse to the University of Rochester School of Medicine in 1962, where he became head of the Department of Clinical Pharmacology. At Rochester, he interacted with William and Margaret Neuman in the Department of Radiation Biology and Biophysics on mineral metabolism, parathyroid hormone, and osteoblast biology. Larry's research career in Rochester was enhanced by a prior experience in the Strangeways Research Laboratory in Cambridge, England. In 1960, he spent a sabbatical year picking up the tricks of organ culture from Dame Honor B. Fell, director of the Laboratory and one of the world's experts in organ culture. There, he was given some bench space and the facilities of the laboratory to use. Hector Deluca first met Larry in 1960 at the Strangeways Laboratory. They arrived within a month of each other as sabbatical faculty. Larry and Hector shared a lab bench, a ½-mL graduated pipette, glassware, and rooster plasma, which was obtained by accompanying Victor, the technician, secretary, and general-purpose manager of the lab, to a hen house in back of the lab where they extracted blood from one of five big roosters. This was excellent training in how to do much with very little, and it prepared both Larry and Hector for later belt tightening when NIH funds became short in supply. Hector recognized early on Larry's inner drive: if it were worth learning, experiencing, or seeing, Larry would be there with boundless energy. Larry's and Hector's paths would cross many times thereafter. On NIH Study Sections, for example, Hector and other members were treated to a man who read every grant (60 per meeting) and was able to comment on them all! While always rigorous, he was also always the defender of young and junior investigators. Larry published two brief, single-authored communications in Nature in 1963. In the first of these papers, he reported the development of a technique for prelabeling embryonic bone rudiments with radiocalcium given to pregnant mothers to establish an assay for direct effects of parathyroid hormone on bone. This approach was subsequently used most successfully in the Raisz lab to understand the regulation of bone resorption. The second of these two short papers on the responses of cultures of parathyroid glands to altering ambient concentrations of mineral ions was also a jewel. It must have taken much hard work to locate, remove, and successfully culture parathyroid glands from 13-day embryonic chicks and analyze cellular changes histologically with bioassays he devised. Kits weren't available then. This work formed the basis for much of the future of the Raisz laboratory's endeavors. Larry's time at Rochester proved to be a period of great scientific growth. Paula Stern came to work with Larry as a postdoctoral fellow in 1963–1964. Larry mentored by example as he worked side-by-side with her in the laboratory, helping to troubleshoot any problems. Every week, he and Paula would go through Current Contents and check off papers of interest. Larry's secretary, Jane Goodale, would collect the journals and set them aside in a room in the library. In the weekly journal club, Larry and Paula would each report on multiple papers and engage in lively discussion. Larry always zeroed in on the critical experiment. He conveyed a sense that science was not about competition, because there were many problems to be solved and many questions to be asked. He would say, “there is enough for everyone.” During the Rochester years, Larry spent another sabbatical at the National Institute of Dental Research, which led to the description of osteoclast activating factor and a paper in Science. While at the Dental Institute, he learned the collagenase digestible protein assay and applied it as a measure of bone collagen production in primary rodent calvarial organ cultures. That seminal work launched the careers of a number of young scientists. Greg Mundy also joined him in Rochester. It was then that prostaglandins assumed an important role in Larry's thinking about bone biology. In 1974, Larry joined the new School of Medicine at the University of Connecticut Health Center in Farmington as the first chief of the Division of Endocrinology and Metabolism. From 1974 to 1997, he was instrumental in building an internationally known program in bone biology with Greg Mundy and new colleagues (or “bone heads,” as he would call them), such as Ernie Canalis, Barbara Kream, Joe Lorenzo, Marja Hurley, Carol Pilbeam, Gideon Rodan, David Rowe, and many others. Eventually, they were able to help him maintain a grasp on the basic science and shift some of his emphasis to clinical osteoporosis with colleagues such as Pam Taxel and Anne Kenny. Always dedicated to basic science and the laboratory (“Show me the data!”), he realized the importance of organizations such as ASBMR and NOF in further advancing awareness of disorders of bone and mineral metabolism. He became the preeminent statesman for the bone field. Marja Hurley was one of the first to work with Larry in Connecticut when she was a UConn medical student in 1974. Larry's mentorship led to her joining the faculty of the UConn and to a deep friendship for the next 30 years. A series of new fellows and new faculty arrived at the Health Center in the late 1970s. Barbara Kream arrived on Larry's doorstep in 1977 looking to complete her last year of postdoctoral training. She initially came to interview with Larry on a sunny Tuesday morning several months before her start in the lab. She was led to a small room where she encountered a masked man with a knife. It was Larry participating in “bone-cutting Tuesday,” when he and the other lab members would dissect the radiolabeled fetal rat long bones for the weekly culture experiments. Around the same time, David Rowe began his career at the Health Center as the head of the Division of Pediatric Endocrinology. Gideon Rodan had arrived as head of Oral Biology at UConn. The remarkable scientific, professional, and personal relationship with Gideon and Sevgi Rodan helped to define an era when the center of the bone universe had clearly tilted in their direction. With gathering scientific momentum, Larry spoke optimistically about the newly formed UConn medical and dental schools that were recruiting extremely accomplished research-oriented faculty in both the basic and clinical sciences and how these programs would be the epicenter of academic medical training for the region. Many hours of planning went on in his second floor office (with its threadbare couch) in which program projects, clinical and basic training grants, and General Clinical Research Center applications were formulated and new research ideas discussed. Joe Lorenzo started with Larry as an endocrine fellow in 1977. Joe quickly recognized that Larry lived for his science and was devoted to understanding biology and applying this knowledge to patient care. Larry was practicing the craft of translational research long before it became fashionable. He believed that discovery would bring us better patient therapies and he worked tirelessly toward this goal. Larry's greatest joy was the time that he spent in the lab, designing experiments and preparing rodent bones for those fabled organ cultures. There was always a special gleam in his eye when he would outline each of the groups in a study, making sure to complete the matrix of control and experimental conditions to include all the “perms and coms” of the conditions that were being tested. Even during his final days, he found a way to design another experiment. Carol Pilbeam first heard Larry speak on calcium and osteoporosis in 1985 during her medical residency. A study of this “new” disease seemed perfect for Carol, an ex-geologist-turned-geriatrician. Larry always had trouble saying no to anyone who wanted to do research, so she arrived in his lab two years later with no research funds and very little laboratory experience. It was some years before she realized just how generous Larry had been in letting an unknown person settle into his lab. Larry was a mentor, colleague, and friend to Carol for more than 23 years. They shared lab space, research projects, students, and transgenic mice, as well as leftovers in the lunchroom. If it had not been for Larry, Carol would never have skied down a hill or fallen off a windsurfer. His positive attitude and enthusiasm kept her and the lab going through the tough times. A gifted scientist who was passionate about his own research, Larry loved to hear and think about the research of others and was apt to make some insightful comment wherever he went. His door was always open to his colleagues and students. He had amazing energy, generosity of spirit, and a phenomenal ability to enjoy so much at one time. Like so many others, Carol always looked forward to seeing him because he made everything more interesting and fun. How many hours were spent in his office with his colleagues, sitting on the threadbare, orange corduroy couch that had the broken leg! One of Larry's strengths was his ability to engage people from all over the world and bring many of them to Connecticut for brief or extended stays. Jean Feyen first met Larry at the European Symposium on Calcified Tissues, held in Angers, France, in 1984. Jean had just started work for his doctoral thesis on the regulation of the production of prostaglandins and bone cells. Reviewing the literature on this topic, it became obvious that Larry's name appeared in most of the articles on this topic. After his first discussion with Larry, Jean realized that spending some time with this remarkable man would be a tremendous opportunity to develop and expand his scientific horizons. Jean remembers Larry's tremendous energy and drive. Despite his challenging work schedule, Larry would always find the time to discuss the progress of the lab work. The danger of these update sessions was that one experimental observation would trigger at least five new hypotheses and 20 new experiments. If time were short during the week, Larry would invite people from the lab to join him on one of his weekend activities, such as sailing or windsurfing on a lake near the Health Center. During these outings, there was always enough time to talk about lab activities. Larry always provided his trainees the opportunities to present work in progress at workshops and meetings and discuss ongoing projects with the many scientists visiting the Raisz laboratory from around the globe. Larry trained many Japanese postdoctoral fellows in bone biology research. The Japanese bone societies are grateful that many of those people, including Toshiyuki Yoneda and Masaki Noda, are now supporting and advancing bone research in Japan because of Larry. He visited Japan frequently to meet his friends Etsuro Ogata and Tatsuo Suda, who were founders of the present Japanese bone societies. Hiroshi Kawaguchi's career as a researcher started with a fateful encounter with Larry in 1991. Hiroshi spent time in the Raisz group from 1991 to 1994, and this experience was formative in his decision to embark on preclinical translational research that would lead to the treatment of skeletal disorders. Larry was his role model as a researcher and mentor. Even after Hiroshi left Connecticut, Larry visited him several times in Japan and kept current of his career. In fact, Hiroshi received an email from Larry's address after he passed away. It was actually was from Larry's wife Helen. However, Hiroshi believed the email was directly from Larry inquiring about the Japanese traditional Obon Week. This is a week during the year when spirits of deceased persons come from heaven to this world in order to meet their family and friends. Hiroshi sent him a reply asking him to visit Japan, since heaven is equidistant from Connecticut and Japan. In 1997, Larry stepped down as head of the Division of Endocrinology and Metabolism and Andrew Arnold was recruited to become director of the Center for Molecular Medicine and division head. Larry played a crucial role in recruiting Andy to Connecticut from Massachusetts General Hospital, and Andy considered it a huge honor to succeed him as division head. As must be true for so many in the bone field, Andy remembers that Larry asked the first and best question from the audience after his first-ever oral presentation at ASBMR. Over the following years, Andy's already tremendous respect and appreciation for Larry's intellect and generative qualities grew and grew. During the next few years at national meetings, Andy would occasionally be asked if Larry had retired, and he could barely control his laughter when responding! Larry, of course, was as busy as ever, taking on new projects and roles both at home and abroad; his energy knew no bounds. ASBMR members can well imagine how amazing it was to have Larry at seminars, research meetings, and clinical conferences. Indeed, Larry's presence transformed the more routine parts of life into something special. The Charge of the White Coat Brigade They come by their hundreds in order to ease The suffering of illness, the pain of disease They teach us all about healthy behavior For in the long run, prevention is saviour. But to maintain the progress of this fine brigade New knowledge must be sought, new discoveries made Government and foundations, which once were there Are finding their cupboards ever more bare. But if we all work and plan together This is a storm we all can weather. And make better health our next port of call. But this is a gala so let's have a ball. Larry Raisz, April 2010 As eloquently expressed by Bill Peck, Larry was the quintessential investigator/mentor; he had a prodigious knowledge of the field, a fertile scientific imagination, boundless energy and enthusiasm, and seemingly limitless patience and listening ability. He was a master communicator —direct, clear, honest, self-effacing, and unfailingly humorous. As a keen student of human nature, he was genuinely interested in us as individuals and as scientists. He enjoyed the successes of his colleagues and trainees. These qualities in rare combination, coupled with his direct, realistic approach, stimulated great confidence among those of us who had the outstanding fortune to learn from and work with him. Larry will always be with all of us in spirit and we will continue to mentor young investigators and do “good science” as exemplified by this remarkable man. Larry Raisz was a friend to many

MicroRNA attenuation of protein translation has emerged as an important regulator of mesenchymal cell differentiation into the osteoblast lineage. A compelling question is the extent to which miR biogenesis is obligatory for bone formation. Here we show conditional deletion of the Dicer enzyme in osteoprogenitors by Col1a1-Cre compromised fetal survival after E14.5. A mechanism was associated with the post-commitment stage of osteoblastogenesis, demonstrated by impaired ECM mineralization and reduced expression of mature osteoblast markers during differentiation of mesenchymal cells of ex vivo deleted Dicerc/c. In contrast, in vivo excision of Dicer by Osteocalcin-Cre in mature osteoblasts generated a viable mouse with a perinatal phenotype of delayed bone mineralization which was resolved by 1 month. However, a second phenotype of significantly increased bone mass developed by 2 months, which continued up to 8 months in long bones and vertebrae, but not calvariae. Cortical bone width and trabecular thickness in DicerΔoc/Δoc was twice that of Dicerc/c controls. Normal cell and tissue organization was observed. Expression of osteoblast and osteoclast markers demonstrated increased coupled activity of both cell types. We propose that Dicer generated miRs are essential for two periods of bone formation, to promote osteoblast differentiation before birth, and control bone accrual in the adult.

We previously demonstrated that parathyroid hormone (PTH) induced interleukin-6 (IL-6) expression in osteoblasts primarily via the cAMP-PKA signaling pathway. In this study, we explored the molecular mechanism(s) underlying this action. We used a fragment of the human IL-6 proximal promoter containing two previously identified, juxtaposed CRE and C/EBP sites at -165/-158 bp (CRE165) and -157/-148 bp (C/EBP157) to drive the expression of a luciferase reporter (IL6-Luc225). Osteoblastic MC3T3-E1 cells were stably and transiently transfected with IL6-Luc225 constructs carrying mutations or deletions of the CRE and C/EBP sites. We demonstrated that agonists that stimulated the cAMP-PKA pathway, also induced IL6-Luc225 activity. The proximal CRE sequence (CRE165) was important in mediating cAMP induction of IL-6 promoter activity, whereas the C/EBP157 did not play a role in this. We also identified a distal CRE-like site (CRE209) that may involve in mediating cAMP-PKA induction of IL-6 promoter activity. Electrophoretic mobility shift assays showed the formation of DNA/protein complexes on the proximal CRE/C/EBP site. We used antibody supershift assays to show the presence of CREM/CREB, phosporylated CREB, C/EBPβ, and C/EBPδ” in the complexes. Our data suggest that PTH-mediated induction of IL-6 promoter activity is regulated primarily by the cAMP-PKA pathway and CRE165 may play a key role in mediating this response.

The goal of this study was to characterize the bone phenotype and molecular alterations in Col3.6-HSD2 mice in which a 3.6-kb Col1a1 promoter fragment drives 11ß-HSD2 expression broadly in the osteoblast lineage to reduce glucocorticoid signaling. Serum corticosterone was unchanged in transgenic females exluding a systemic effect of the transgene. Adult transgenic mice showed reduced vertebral trabecular bone volume and reduced femoral and tibial sub-periosteal and sub-endosteal areas as assessed by microCT. In adult female transgenic mice, histomorphometry showed that vertebral bone mass and trabecular number were reduced but that osteoblast and osteoclast numbers and the mineral apposition and bone formation rates were not changed, suggesting a possible developmental defect in the formation of trabeculae. In a small sample of male mice, osteoblast number and percent osteoid surface were increased but the mineral apposition bone formation rates were not changed, indicating subtle sex-specific phenotypic differences in Col3.6-HSD2 bone. Serum from transgenic mice had decreased levels of the C-terminal telopeptide of α1(I) collagen but increased levels of osteocalcin. Transgenic calvarial osteoblast and bone marrow stromal cultures showed decreased alkaline phosphatase and mineral staining, reduced levels of Col1a1, bone sialoprotein and osteocalcin mRNA expression, and decreased cell growth and proliferation. Transgenic bone marrow cultures treated with RANKL and M-CSF showed greater osteoclast formation; however, osteoclast activity as assessed by resorption of a calcium phosphate substrate was decreased in transgenic cultures. Gene profiling of cultured calvarial osteoblasts enriched in the Col3.6-HSD2 transgene showed modest but significant changes in gene expression, particularly in cell cycle and integrin genes. In summary, Col3.6-HSD2 mice showed a low bone mass phenotype, with decreased ex vivo osteogenesis. These data further strengthen the concept that endogenous glucocorticoid signaling is required for optimal bone mass acquisition and highlight the complexities of glucocorticoid signaling in bone cell lineages.

Background: Abolishing the inhibitory signal of intracellular cAMP by phosphodiesterases (PDEs) is a prerequisite for effector T (Teff) cell function. While PDE4 plays a prominent role, its control of cAMP levels in Teff cells is not exclusive. T cell activation has been shown to induce PDE8, a PDE isoform with 40- to 100-fold greater affinity for cAMP than PDE4. Thus, we postulated that PDE8 is an important regulator of Teff cell functions.Methodology/Principal Findings: We found that Teff cells express PDE8 in vivo. Inhibition of PDE8 by the PDE inhibitor dipyridamole (DP) activates cAMP signaling and suppresses two major integrins involved in Teff cell adhesion. Accordingly, DP as well as the novel PDE8-selective inhibitor PF-4957325-00 suppress firm attachment of Teff cells to endothelial cells. Analysis of downstream signaling shows that DP suppresses proliferation and cytokine expression of Teff cells from Crem(-/-) mice lacking the inducible cAMP early repressor (ICER). Importantly, endothelial cells also express PDE8. DP treatment decreases vascular adhesion molecule and chemokine expression, while upregulating the tight junction molecule claudin-5. In vivo, DP reduces CXCL12 gene expression as determined by in situ probing of the mouse microvasculature by cell-selective laser-capture microdissection.Conclusion/Significance: Collectively, our data identify PDE8 as a novel target for suppression of Teff cell functions, including adhesion to endothelial cells.

Murine MC3T3-E1 and MC-4 cells were stably transfected with -371/+70 bp of the murine cyclooxygenase-2 (COX-2) promoter fused to a luciferase reporter (Pluc371) or with Pluc371 carrying site-directed mutations. Mutations were made in (1) the cAMP response element (CRE) at -57/-52 bp, (2) the activating protein-1 (AP-1)-binding site at -69/-63 bp, (3) the nuclear factor of activated T-cells (NFAT)-binding site at -77/-73 bp, and (4) both the AP-1 and NFAT sites, which comprise a composite consensus sequence for NFAT/AP-1. Single mutation of CRE, AP-1, or NFAT sites decreased parathyroid hormone (PTH)-stimulated COX-2 promoter activity 40% to 60%, whereas joint mutation of NFAT and AP-1 abrogated the induction. On electrophoretic mobility shift analysis, PTH stimulated binding of phosphorylated CREB to an oligonucleotide spanning the CRE and binding of NFATc1, c-Fos, and c-Jun to an oligonucleotide spanning the NFAT/AP-1 composite site. Mutation of the NFAT site was less effective than mutation of the AP-1 site in competing binding to the composite element, suggesting that cooperative interactions of NFATc1 and AP-1 are more dependent on NFAT than on AP-1. Both PTH and forskolin, an activator of adenylyl cyclase, stimulated NFATc1 nuclear translocation. PTH- and forskolin-stimulated COX-2 promoter activity was inhibited 56% to 80% by calcium chelation or calcineurin inhibitors and 60% to 98% by protein kinase A (PKA) inhibitors. These results indicate an important role for the calcium-calcineurin-NFAT signaling pathway in the PTH induction of COX-2 and suggest that cross-talk between the cAMP/PKA pathway and the calcium-calcineurin-NFAT pathway may play a role in other functions of PTH in osteoblasts.

During the phase of overt tooth cytodifferentiation that occurs after birth in the mouse and using the 3.6Collagen1a-Cre and the BMP4 floxed and BMP4 knockout mice, the BMP4 gene was deleted in early collagen producing odontoblasts around postnatal day 1. BMP4 expression was reduced over 90% in alveolar osteoblasts and odontoblasts. There was decreased rate of predentin to dentin formation and decreased mature odontoblast differentiation reflected in reduced DMP1 expression and proper dentinal tubule formation, as well as reduced Collagen type I and Osteocalcin expression. We observed mutant dysmorphogenic odontoblasts that failed to properly elongate and differentiate. The consequence of this failed differentiation process leads to permanent loss of dentin thickness, apparent enlarged pulp chambers in the molars and reduced bone supporting the tooth structures in mice as old as 10–12months. Deletion of the BMP4 gene in odontoblasts also indirectly disrupted the process of enamel formation that persisted throughout life. The mechanism for this altered differentiation program in the absence of the BMP4 gene in odontoblasts is from decreased BMP signaling, and decreased expression of three key transcription factors, Dlx3, Dlx5, and Osterix. BMP signaling, as well as Dlx3 and Amelogenin expression, is also indirectly reduced in the ameloblasts of the odontoblast BMP4 cKO mice. This supports a key paracrine or endocrine postnatal role of odontoblast derived BMP4 on the proper amelogenesis and formation of the enamel.

Because global deletion of the prostaglandin EP4 receptor results in neonatal lethality, we generated a mouse with targeted EP4 receptor deletion using Cre-LoxP methodology and a 2.3 kb collagen I a1 promoter driving Cre recombinase that is selective for osteoblastic cells. We compared wild type (WT), global heterozygote (G-HET), targeted heterozygote (T-HET) and knockout (KO) mice. KO mice had one targeted and one global deletion of the EP4 receptor. All mice were in a mixed background of C57BL/6 and CD-1. Although there were one third fewer G-HET or KO mice at weaning compared to WT and T-HET mice, G-HET and KO mice appeared healthy. In cultures of calvarial osteoblasts, prostaglandin E-2 (PGE(2)) increased alkaline phosphatase (ALP) activity in cells from WT mice, and this effect was significantly decreased in cells from either G-HET or T-HET mice and further decreased in cells from KO mice A. selective agonist for EP4 receptor increased ALP activity and osteocalcin mRNA levels in cells from WT but not KO mice. A selective COX-2 inhibitor, NS-398, decreased osteoblast differentiation in WT but not KO cells. At 15 to 18 months of age there were no differences in serum creatinine, Calcium, PTH, body weight or bone mineral density among the different genotypes. Static and dynamic histomorphometry showed no consistent changes in bone volume or bone formation. We conclude that expression of the EP4 receptor in osteoblasts is critical for anabolic responses to PGE(2) in cell culture but may not be essential for maintenance of bone remodeling in vivo. (C) 2009 Elsevier Inc. All rights reserved.

ICER is a member of the CREM family of basic leucine zipper transcription factors that acts as a dominant negative regulator of gene transcription. Four different isoforms of ICER (I, Igamma, II and IIgamma) are transcribed from the P2 promoter of the Crem gene. We previously found that each of the ICER isoforms is induced by parathyroid hormone in osteoblasts. The goal of the present study was to assess the function of ICER in bone by overexpressing ICER in osteoblasts of transgenic mice. ICER I and ICER II cDNAs, each containing an N-terminal FLAG epitope tag, were cloned downstream of a fragment containing 3.6 kb of the rat Col1a1 promoter and most of the rat Col1a1 first intron to produce pOBCol3.6-ICER I and pOBCol3.6-ICER II transgenes, respectively. Multiple lines of mice were generated bearing the ICER I and ICER II transgenes. At 8 weeks of age, ICER I and ICER II transgenic mice had lower body weights and decreased bone mineral density of femurs and vertebrae. Further studies were done with ICER I transgenic mice, which had greatly reduced trabecular bone volume and a markedly decreased bone formation rate in femurs. Osteoblast differentiation and osteocalcin expression were reduced in ex vivo bone marrow cultures from ICER I transgenic mice. ICER I antagonized the activity of ATF4 at its consensus DNA binding site in the osteocalcin promoter in vitro. Thus, transgenic mice with osteoblast-targeted overexpression of ICER exhibited osteopenia caused primarily by reduced bone formation. We speculate that ICER regulates the activity and/or expression of ATF/CREB factors required for normal bone formation.