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Osteo-Odyssey: A Memoir Paula H. Stern Department of Molecular Pharmacology and Biological Chemistry, Northwestern University, Feinberg School of Medicine, Chicago, IL, USA

STARTING OUT: FROM A TO B ONE

Ultimately, it led me to become involved in the organizations upon which science is dependent, including professional organizations, federal agencies, and foundations involving interactions between medical professionals and lay groups.

Almost 50 years ago, in the fall of 1963, I entered the bone field when I became Larry Raisz’s first Ph.D. postdoctoral fellow. It was not because I had been interested previously in osteoporosis or even in bone. In college, I had particularly enjoyed invertebrate zoology. Not a good predictor of a career in bone research. My Ph.D. in pharmacology from the University of Michigan was on carbon tetrachloride hepatotoxicity, examining effects on oxidative phosphorylation and triglyceride metabolism. Again not very bone related. But I did have a continuing thread of interest in endocrinology, writing my M.S. qualifying exam on thyroid hormone analogs, taking an experimental endocrinology elective as a graduate student, being intrigued by the permissive effect of adrenocortical steroids on the fatty liver elicited by carbon tetrachloride, and going to Woods Hole (MA, US) for the comparative endocrinology course. I decided to seek postdoctoral training in endocrine pharmacology, which gave me enough latitude, as one-half of a dual career couple, to explore multiple possibilities. One of my postdoc interviews was at a Federation of American Societies for Experimental Biology (FASEB) meeting in Atlantic City, where the representative of the pharmacology department at the University of Rochester was Larry Raisz, a physician who had recently joined their department to establish a clinical pharmacology program. I was intrigued by Larry’s enthusiasm, and decided it would be interesting to learn more about parathyroid hormone (PTH) and bone. I was in Larry’s laboratory for less than 1 year, again due to dual-careercouple factors, but the months there ensnared me into a rapidly expanding field that presented many interesting questions. It also provided me with the opportunity to interact with the leaders of the field as they became ascendant, and with bright and dedicated young investigators from the many areas that converged on bone.

Osteoporosis. http://dx.doi.org/10.1016/B978-0-12-415853-5.00005-4

BEING THE FIRST POSTDOC IN LARRY’S LAB Larry’s space at the University of Rochester at that time was small. His office adjoined a tiny laboratory with a culture hood and carbon dioxide (CO2) incubator, a fraction collector and columns, and benches for histology and other laboratory work. We cultured parathyroid glands and bones. Every Tuesday morning we would dissect radii and ulnae of fetuses from 45Ca-prelabeled pregnant rats, and study effects of treatments. Sometimes two of us, which often included Larry, would be dissecting at the same time. With protein purification, histology, and general laboratory work all going on in the same area, it is amazing that the cultures did not get contaminated, but somehow it worked. We would meet and plan experiments in his office, or in the medical library where we would reserve a room and present and discuss papers we had selected from that week’s Current Contents. The group was small: Larry, Bill Au, a young physician whom Larry had recruited to the clinical pharmacology program, and Alan Poland, a medical student who was taking one year off to do research. The strong clinical backgrounds of these other members of the group gave me an awareness of bone physiology and pathophysiology, including osteoporosis, and made future collaborations with other clinicians easy to undertake. After leaving Rochester, Bill Au has had a highly successful career in endocrine clinical pharmacology in both academia and industry. Alan Poland was greatly inspired to do basic research by his year

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in Larry’s laboratory, and later, as a faculty member at the McArdle Institute at the University of Wisconsin, made seminal discoveries on the mechanism of dioxin toxicity leading to the recognition of the aryl hydrocarbon receptor. Alan subsequently became the Program Director of the Cancer Etiology Branch of the National Cancer Institute. In Larry’s laboratory, I worked with Bill and Alan on the regulation of PTH secretion in vitro [1], and also began studies on the components of serum required for the in vitro effect of PTH on bone [2], which led to later studies from my own laboratory that described a bone-resorbing factor in the albumin fraction of bone [3,4]. From my brief postdoctoral period, I acquired an interest in any research involving bone, an enjoyment of discussing experimental results, both from anyone’s laboratory and from the literature, and a sustained connection with Larry’s laboratory. I visited a number of times over the years and consider many of the subsequent members of the laboratory as members of my extended scientific family.

IN THE ACADEMIC WORLD: PROJECTS AND PEOPLE After the unusually brief postdoctoral stint, my husband Robert and I returned to the University of Michigan for two years, where he pursued his studies in literature and I became an instructor in the Pharmacology department. While at Michigan, I prepared my first National Institutes of Health (NIH) grant application on “Effects of Hormones on Bone Resorption in vitro”, and, in those days of good funding, I was able to obtain funding on my first attempt, and have a grant in 1966 when I started as an Assistant Professor at Northwestern University. I had no idea how fortunate I was, as this was prior to the time when the Association for Women in Science filed a suit against the Department of Health and Human ­Services (HHS) because the underrepresentation of women on Study Sections was depriving women of fair reviews of their grant proposals. I do not know who the members of the Study Section reviewing bone grants in 1965 were, but they obviously were supportive of women.

Models At Northwestern, I set up the fetal rat limb bone organ culture system. Over the next several years it was to provide us with interesting observations. Its application in work on vitamin D analogs and on thyroid hormone provided clinically useful information. Other studies, on glucocorticoids and on calcineurin inhibitors, demonstrated that isolated systems can give results that need to be reconciled with the total physiologic response. A particularly appealing aspect of the fetal rat limb bone organ culture system was its amenability to quantitative

studies. Calculation of % 45Ca release provided a useful parameter for comparative data. As a pharmacologist, it was important for me to be able to generate doseresponse curves, and the model provided that capability. Organ culture also had the appeal of being architecturally intact, yet isolated from multiple systemic factors, allowing “direct” effects to be examined. We were also interested in visualizing the cellular responses in these tiny millimeter-long tissues. The increase in 45Ca release was reflected in an increase in multinucleated osteoclasts, as was shown by a heroic, labor-intensive study carried out by Tom Hefley, in which he isolated intact osteoclasts with hyaluronidase and tediously counted the number of detectable nuclei [5] (Fig. 5.1). Over the years, we introduced some additional bone organ culture models that had different attractive features. A dentist in the Masters in Dental Research program, Jim Parkhill, cultured fetal rat mandibles and examined their responses to PTH and interleukin (IL)-1β [6]. Vertebral bone cultures were explored by Pamela Stewart, a postdoctoral fellow. We found that these bones resorbed even more quickly and dramatically than the limb bones [7] (Fig. 5.2). The vertebral dissections were very tedious and the model was not exploited beyond that publication. In contrast, neonatal mouse calvarial cultures, which Nancy Krieger introduced when she joined the laboratory, were a useful model that could be mastered more quickly than the limb bone cultures. The calvaria gave quantitative results comparable to the limb bones [8] and being larger, did not require 45Ca labeling but allowed resorption to be determined from the calcium released into the medium. A further modification of the calvarial model was a method for measuring resorption and collagen synthesis simultaneously in the cultures [9]. Nancy Krieger and Tom Hefley carried out many other interesting studies and became junior faculty members in the department before moving on, Nancy to a faculty position at the University of Rochester, and Tom to designing computer systems for hospital laboratories. Stuart Stock, a materials scientist at Northwestern applied micro-computerized tomography (micro-CT) to evaluating resorption in the calvarial cultures. It was gratifying to see that the F value, the fraction resorbed, showed a good correlation with the calcium released [10] (Fig. 5.3).

Vitamin D and Initial Collaborations When I first arrived at Northwestern, I looked for other bone researchers with whom I might interact and was fortunate to find an interested colleague in Norman Bell, who was at that time in the Endocrinology Division of the Department of Medicine. We began a collaboration that was to extend over several years, even after Norman moved to South Carolina. Our initial collaborative studies were on (thyro)calcitonin [11,12] and glucagon [13],

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FIGURE 5.1  Frequency distribution of the number of nuclei per osteoclast in cultured fetal rat limb bones. The cells were obtained by hyaluronidase treatment of bones cultured for 72 hours. Upper figure left: uncultured bones; upper figure right: cultured control bones; lower figure left: cultured bones treated with 10 pM calcitriol, with a ratio of 45Ca release from treated/control of 2.02; lower figure right: osteoclasts isolated from cultured bones. Source: Hefley and Stern (1982) [5].

in rats and in organ cultures. We found that glucagon had hypocalcemic effects and inhibited bone resorption in vitro. Other studies confirmed antiresorptive and antiosteoporotic effects of glucagon [14–16], but its other systemic effects actions precluded its application as a therapeutic agent for bone. It is interesting that studies of enteric hormones have identified the related gut hormones, glucagon-like peptides, as having a physiologic role in skeletal metabolism [17,18] and showing therapeutic effectiveness in the treatment of the osteopenia following distal ileostomy [19]. The major work that resulted from the collaboration with Norman Bell, sometimes together with other investigators including Sol Epstein, Hector DeLuca, Russell Turner, Phil Lambert, and Susan Paulson, were clinical studies related to the vitamin D metabolite calcitriol, for which we used the fetal rat limb bone system as a bioassay. With a series of purification steps, it was possible to isolate calcitriol from serum and bioassay it with the organ cultures of fetal bones. The purification was carried out by two alternative procedures. The first procedure involved a collaboration with Hector DeLuca, from the nearby University of Wisconsin. Hector generously invited me to spend two summer sabbaticals in his laboratory where I learned from Yoko Tanaka, his research associate, and Mike Holick, then an MD/PhD student,

the chromatographic techniques they were using to isolate vitamin D metabolites from serum. The procedure involved extraction with dichloromethane, chromatography on Sephadex LH-20, and purification on silicic acid by high-performance liquid chromatography (HPLC) [20]. Recovery was monitored by addition of 26,27-[3H]-calcitriol to the plasma. Back at Northwestern University, we devised a second procedure in which we first extracted the serum with benzene or isopropyl ether, then did a back extraction with 0.1 M Na2HPO4, followed by the final HPLC purification step [21]. The cultures were highly sensitive to calcitriol (Fig. 5.4), and could detect 1  pg of the metabolite, which was lower than the detection limits of the radioreceptor assays being used at the time we developed the assay. Reproducibility was good and serum from patients with various conditions provided consistent results (Fig. 5.4). Application of the bioassay allowed us to make a number of important clinical observations. The studies revealed elevated calcitriol in vitamin D-dependent rickets type II, suggesting end-organ resistance [22]. The risk of hypercalcemia with calcitriol use was shown by the association of elevated calcitriol with hypercalcemic episodes observed with long-term treatment with the analog [23,24] (Fig. 5.5). Other studies revealed a role of excessive calcitriol production in the increased sensitivity

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FIGURE 5.2  Parathyroid hormone (PTH) elicits rapid and extensive resorption in vertebral bones. Scanning electron micrograph of control and PTH-treated vertebrae. Neonatal (4–6 days) mouse vertebral bones (vertebrae #12 – #15 from the tip of the tail) were cultured in the presence or absence of 10 nM PTH for 24, 48, 72, or 96 hour. The arrows indicate the border of the area covered by cells that have migrated out of the bone tissue. Magnification = 40×. Source: Stewart and Stern (1987) [7].

to vitamin D in patients with sarcoidosis and patients with tuberculosis [25–27]. The existence of extrarenal sites of calcitriol production was confirmed in anephric patients [28], a study in which serum samples were split and analyzed by both a radioreceptor assay and the bioassay. The results revealed remarkable correlation. Other studies on the regulation of calcitriol in children showed differences between black and white children [29,30]. Although the bioassay method was labor intensive, the findings were valuable and the collaborations enjoyable. However, once sensitive radioimmunoassays and radioreceptor assays became widely available, the bioassay was no longer needed. Another aspect of the collaboration with Hector DeLuca was a series of studies on the in vitro effects of

vitamin D analogs on bone. Hector and his colleagues had synthesized a number of interesting analogs, and the bone organ culture system allowed us to compare activity on bone with activity on the intestine, which was being studied in Hector’s laboratory. In addition, comparison of in vitro studies with receptor binding studies and in vivo findings could indicate the importance of pharmacokinetic factors in differential activity of compounds. The collaboration was exciting and productive, resulting in a number of papers on analogs [31–40]. We also collaborated with other investigators who had made interesting analogs [41,42]. None of these interesting compounds was more potent than calcitriol in the organ cultures, although some had shown more potency on effects in vivo, suggesting the importance of

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on Analogs” [43]. The interpretation may be more complex than concluded at that time. Glen Jones has pointed out that transactivation activity deriving from a number of other factors may be a more critical determinant of analog potencies than vitamin D receptor binding affinity [44]. Around the time when the analog papers were being published, Paul Munson invited me to write a review on “The D Vitamins and Bone” for Pharmacological Reviews [45]. I cannot recall anything that I have done over the years that was more work than preparing that 34-page, 684-citation, single-authored manuscript! The collaboration with Norman Bell continued after he left Northwestern. I regret that he left, as did several other faculty members with whom I enjoyed collaborating while they were at the university: David Feldman, Jim Bordeau, Laird Madison, Jeremy Gilbert, and Neil Clipstone. Fortunately, other bone scientists came and stayed and became valued colleagues and at times ­collaborators, including Craig Langman, Stuart Sprague, Stuart Stock, Arthur Veis, Tom Schnitzer, Nalini Rajamannan, Andy Bunta, and Beatrice Edwards. I migrated away from vitamin D research as other questions came to dominant the time of the laboratory. However, we did not abandon it entirely. Together with David U’Prichard, a colleague in the pharmacology department, and a graduate student, Harris (Handy) Gelbard, we reported on calcitriol receptors in the pituitary [46] including some evidence of dopaminergic regulation [47], and, in an abstract, on calcitriol receptors in bovine brain and neuronal cell lines [48]. More recently, Debra Dossing, a Ph.D. student, made the interesting observation that the endogenous receptor activator of nuclear factor-kappaB ligand (RANKL) product expressed by UMR-106 cells in response to calcitriol treatment has a molecular weight of 32 kDa, whereas that expressed after treatment with PTH or forskolin (FSK) had a molecular weight of 52 kDa [49]. The two RANKL products interacted differently with osteoprotegerin (OPG). I hope sometime to explore this intriguing finding further.

FIGURE 5.3  Micro-computerized tomography (micro-CT) scan of neonatal mouse calvarial cultures; upper: micro-CT-derived three-­ dimensional renderings of calvaria from A) control; B) 0.3 nM interleukin (IL)-1; C) 1 nM IL-1. Lower: correlation of calcium release with F (fraction resorbed by micro-CT analysis) in two experiments. Source: Stock et al. (2004) [10].

pharmacokinetic factors. There was a striking correlation between the in vitro bone resorbing potencies and the intestinal vitamin D receptor binding potencies reported by a number of laboratories (r = 0.936; Fig. 5.6), which I described in a perspective article entitled “A Monolog

Glucocorticoids, Calcineurin Inhibitors, Selective Estrogen Receptor Modulators and the Expansion of the Laboratory Group Glucocorticoids My first independent publication from my position in pharmacology at Northwestern was a study on the effects of various steroids on PTH-induced 45Ca release in the fetal rat limb bones [50]. In the experiments, gonadal steroids had little effects until micromolar concentrations were used. Interestingly, glucocorticoids inhibited resorption at concentrations above 1  nM, with no significant effects at lower concentrations. I speculated

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FIGURE 5.4  Upper: dose-response of effects of calcitriol on release of 45Ca from cultured fetal rat limb bones. Lower: calcitriol concentrations in serum of healthy individuals and patients with disorders of calcium metabolism, as measured by the organ culture bioassay. NORM, normal; ANEPH, anephric; RF, renal failure; HYPERP, hyperparathyroid; PHP, pseudohypoparathyroid; SARC, sarcoidosis. Source: Stern et al. (1980) [21].

in the discussion that the failure to elicit the expected bone loss that was seen in patients treated with glucocorticoids might be a consequence of the short duration of the study (72  hr) or that the bone loss associated with glucocorticoids might be related to effects on bone formation [50]. In a later collaboration with David Feldman, while he was at Northwestern before moving to Stanford, and Rosemary Dziak, who was a postdoctoral fellow in the laboratory before becoming a faculty member at the State University of New York (SUNY) Buffalo, we demonstrated high affinity glucocorticoid binding in osteoblasts isolated from neonatal mouse calvaria [51]. Glucocorticoid treatment inhibited protein synthesis in the cells [52], consistent with earlier findings by Bill

Peck [53]. Glucocorticoid inhibition of osteoblast activity is now well established, both the specific downregulation of type I collagen [54] and other genes reflecting osteoblast differentiation [55] and promotion of apoptosis of the cells [56]. Osteoblasts are also likely targets for the effect of glucocorticoids to increase osteoclastogenesis, with potential factors being decreased OPG [57], possibly mediated through soluble glucocorticoid-induced tumor necrosis factor (TNF) receptor [58], and increased soluble IL-6 receptor alpha [59]. In an osteoclastogenesis model, glucocorticoid treatment inhibited formation of multinucleated osteoclasts through downregulation of beta3 integrin expression [60], and inhibitory effects of cortisol on rat bone osteolysis have been described [14].

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FIGURE 5.5  Association of elevated calcitriol, measured by the organ culture bioassay, with hypercalcemic episodes, in patients treated with calcitriol. Source: Bell and Stern (1978) [24].

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Glucocorticoids have been found to increase osteoclast life span [61]. A fairly tight dose–response relationship has been found in effects of glucocorticoids on human osteoblast generation and pit-forming activity [62]. Reflecting back on our results in view of these newer findings, it is possible that a resorptive effect might have been detected in the early organ cultures had we used an expanded dose–response curve at the lower glucocorticoid concentrations or have maintained the cultures longer. We still do not know all of the answers.

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Calcineurin Inhibitors Interestingly, similar to the inhibitory effects of the glucocorticoids on stimulated resorption in the bone organ cultures, the calcineurin inhibitors inhibited stimulated resorption in both limb bone organ cultures and calvarial cultures. Our studies were initiated after a pediatric endocrinologist at Northwestern, Orville Green, called me about a patient of his who developed hypocalcemia when treated with cyclosporine. The finding [63] led us to examine the effects of cyclosporine and FK506 on fetal rat limb bone resorption in vitro. We observed that immunosuppressant cyclosporines inhibited resorption mediated by a number of stimulators, including PTH, calcitriol, prostaglandin E2 and osteoclast-activating factor [64]. The effect was not associated with cytotoxicity and could be overcome by higher concentrations of the stimulators of resorption. Cyclosporine also inhibited the resorption elicited by thyroid hormone [65]. In collaboration with the laboratory of Klaus Klaushofer in Vienna, we confirmed the broad and consistent inhibitory effects of cyclosporine on resorption, using the mouse calvaria culture system [66] (Fig. 5.7). Klaus’ paper was awarded the 1987 Austrotransplantpreis. Orcel et al. showed similar results in organ cultures and provided evidence that cyclosporine inhibited osteoclastogenesis, especially on the fusion process [67]. The in vitro findings were consistent with in vivo work by del Pozo et al. [68] revealing that cyclosporine attenuated the bone and cartilage loss in an adjuvant arthritis model. It was, therefore, quite an unexpected finding when Sol Epstein and his colleagues showed that in rats, cyclosporine caused high turnover bone loss [69]. In addition, the clinical findings in patients receiving organ transplants suggested that cyclosporine, as well as glucocorticoids, caused bone loss in the patients [70,71]. As understanding of the pathway has increased, the mechanism of the bone loss has become even more complex. The role and mechanism of the calcineurin-regulated transcription factor nuclear factor of activated T cells (NFAT) in RANKL-mediated osteoclastogenesis was elucidated by Takayanagi and his colleagues [72]. We also had been working on the role of the calcineurin-NFAT pathway in osteoclastogenesis in collaboration with Neil Clipstone, at that time a faculty member in our

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FIGURE 5.7  Inhibition of parathyroid hormone (PTH) (10 nM)stimulated resorption of neonatal mouse calvaria by cyclosporine A (CsA). Significant (p < 0.001) inhibition was obtained with CsA concentrations of 10–7 M and higher. Source: Klaushofer et al. (1987) [66].

Microbiology/Immunology department. From the time Neil came to Northwestern, we kept telling each other that we should follow up on the findings of inhibition in the bone organ cultures with studies on the pathway. The lead on our work was taken by Hiroaki Hirotani, an oral surgeon fellow from Tohoku University, who demonstrated that the immunosuppressants, cyclosporine A and FK506, or the retrovirally mediated expression of a specific calcineurin inhibitory peptide, VIVIT, all inhibited RANKL-induced differentiation of RAW 264.7 cells into mature osteoclasts [73] (Fig. 5.8). Further, ectopic expression of a constitutively active NFATc1 mutant in the cells induced them to differentiate into functional osteoclasts, with relevant effects on gene expression and morphology [73] (Fig. 5.8). In other work, Kaoru Igarashi, also from Tohoku University, and others in the laboratory showed that cyclosporine and FK506 elicited apoptosis in mouse bone marrow cultures [74] (Fig. 5.8). Thus, the direct effects on osteoclastogenesis were consistent with the inhibitory effects first observed in the

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FIGURE 5.8  Upper: the calcineurin inhibitory peptide VIVIT prevents receptor activator of NF-kappaB ligand (RANKL)-stimulated osteoclastogenesis in cultures of RAW 264.7 cells. Middle: constitutively active NFATc1 elicits osteoclastogenesis in cultures of RAW 264.7 cells. Source: reproduced with permission from Hirotani, H. Touhy, N., Woo, J. T., Stern, P. H., Clipstone, N. (2004). The calcineurin/NFAT signaling pathway regulates osteoclastogenesis in RAW 264.7 cells. J. Biol. Chem. 279(14), 13984–13992. © the American Society for Biochemistry and Molecular Biology [73]. Lower: the calcineurin inhibitors cyclosporine A and FK506 elicit apoptosis of osteoclasts derived from peripheral blood mononuclear cells. (See color plate.) Source: Igarashi et al. (2004) [74].

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organ cultures. However, as with the glucocorticoids, work on osteoblasts, both studies on cell proliferation [75,76] and on calcineurin–NFAT signaling in osteoblasts [77–79] suggested that the bone loss elicited by the calcineurin inhibitors could be due to suppression of anabolic effects in osteoblasts. Interestingly, other work has suggested that there are biphasic effects of cyclosporine on osteoblasts and at some concentrations, it could preserve bone [80]. Clearly, both glucocorticoids and calcineurin inhibitors have complex effects on bone and it has been satisfying to have contributed to the story. Selective Estrogen Receptor Modulators In work carried out in the 1980s, we investigated the effects of selective estrogen receptor modulators (SERMS) on bone resorption in vitro. As I recall, I was intrigued by an abstract presented at the Federation Meeting that was followed by a paper from the same group [81] reporting that clomiphene prevented ovariectomy-induced bone loss, and decided to test the drug in the organ culture model. Our results showed that clomiphene and tamoxifen both prevented bone resorption in organ cultures [82]. After encountering the inconsistencies between in vitro and in vivo effects with the glucocorticoids and calcineurin inhibitors immunosuppressants, it was reassuring that this effect was replicated in the in vitro model. It was also exciting to read the papers showing a protective effect, or at least no bone loss, in patients with breast cancer who were being treated with tamoxifen [83,84]. However, it was puzzling as to why 17β-estradiol had not elicited the same dramatic inhibitory effects in the organ cultures in the study described earlier [50]. It is possible that the inhibitory effects of the SERMS on resorption in vitro could be mediated by a mechanism independent of the estrogen receptor.

Thyroid, Endothelin, and Diurnal Rhythms: Trainees and Visitors from Abroad Around 1990, trainees and visiting scientists from overseas with professional degrees in medicine or dentistry began to come to the laboratory. The result was that some of the work was followed up by clinical studies. I have grouped three such topics in this next section. The thyroid hormone studies were the most extensive, but the others were also of interest and were being pursued at the same time. THYROID HORMONES AND BONE

Figure 5.9 shows the results of a PubMed search from December 2011 of endocrine and bone topics. Thyroid hormone is at the bottom of the list. I found this surprising, perhaps biased by my own interest, but also considering the long history, importance, and interesting questions that characterize the topic. The history goes back more than a century. In 1891, more than 120 years ago, von Recklinghausen reported on the bone loss in hyperthyroidism [85]. Thyroid hormone deficiency or resistance impairs skeletal growth [86]. Like PTH, thyroid hormone is capable of both anabolic and resorptive actions (reviewed in [87]). Many interesting aspects of thyroid hormone action on bone have only begun to be explored, such as why younger individuals appear to express the anabolic effects to concentrations that would cause bone loss in older people [88,89], why bone at different sites differs in its responsiveness to thyroid hormone [90,91], and what the paracrine factors are that mediate effects of thyroid hormones. There is an extensive literature of conflicting findings on whether suppressive and even replacement therapy with thyroid hormones can lead to bone loss (reviewed in [87]). The relative roles of thyroid hormone and thyroid-­stimulating hormone (TSH) on bone are being debated [92,93]. The topic is clearly important to both normal physiology and disease.

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FIGURE 5.9  Numbers of PubMed citations on topics of hormones and bone. December 2011. PTH: parathyroid hormone.

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as well as on other topics that will be described. We found rapid (0.5 min) nongenomic effects of thyroid hormones on the inositol phosphate second messenger system [97]. Peter led a project that revealed that thyroid hormones increased insulin-like growth factor I (IGF-I) in bone organ cultures and in osteoblast cell lines [98] (Fig. 5.10). This work was followed by studies on the role of IGF-I in thyroid hormone action on bone. Bill Huang, a Northwestern University medical student, who was taking one year off to work in my laboratory on a Howard Hughes fellowship, worked with Gabor Tarjan, another visiting physician-scientist from Semmelweis University, Laird Madison, in our Endocrine Division, and a dental student, Larry Golden, to determine the bone effects of antagonizing IGF- I with antisense oligonucleotides, neutralizing antibodies to the IGF receptor, and an antagonist peptide. The studies

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If there was a lack of interest in thyroid hormones, it was not reflected in the activity in my laboratory, where it engendered a continuous thread of research. Thyroid hormone receptors were characterized in bone by Nancy Krieger [94], who also carried out studies on the cardiovascular agent milrinone, for which structural homology to thyroid hormone had been reported. The studies concluded that the milrinone effects were probably independent of the thyroid hormone receptor [95]. Two endocrinologists, Guo-guang Du, in my laboratory, and his wife Wen-Xia Gu, in the Endocrine division, showed a mutual upregulation between thyroid hormone receptors and PTH receptors in ROS 17/2.8 osteoblastic cells [96]. With Peter Lakatos, who came to my laboratory as a young physician-scientist fellow from Semmelweis University in Hungary, we carried out a number of interesting studies on thyroid hormone

Nonsense

FIGURE 5.10  Upper: T3 increases insulin-like growth factor-I (IGF-I) in medium from cultures of (left) UMR-106 cells and (right) fetal rat limb bones. T3 effects were significant (p < 0.05) at 10–10 M and reached a peak at 10–8 M. UMR-106 cells were treated for 48 hours, and limb bones were treated for 72 hours. Source: Lakatos et al. (1993) [98]. Lower: T3 (10–8 M) stimulates alkaline phosphatase activity in MC3T3-E1 cells, and transfection with IGF-I receptor antisense oligonucleotide blocks the response. Values are means ± standard error of the mean. + p < 0.05; ** p < 0.01. Source: Huang et al. (2000) [99].

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showed that these antagonists prevented thyroid hormone effects on osteoblast cell proliferation, alkaline phosphatase (Fig. 5.10), osteocalcin, and proline incorporation [99]. The publication, in the Journal of Bone and Mineral Research, in addition to contributing to our understanding of the mechanism of the anabolic effects of thyroid hormones, was regarded as a very positive component of Bill’s application for his orthopedic surgery residency. The tools that were acquired for the studies of the role of IGF in thyroid hormone responses also proved useful in later studies on bone anabolic effects of prostate cancer cell conditioned media, which were shown by a student, Rumi Bhattacharyya, to be mediated through IGF-I and the mitogen-activated protein (MAP) kinase pathway [100]. After Peter Lakatos returned to Hungary, we obtained a Fogarty International Center grant and established a collaboration with Subburaman Mohan and David Baylink to ­ examine IGF-I and IGF binding proteins (IGFBP) in patients with hyperthyroidism. The patients showed increases in IGF-I, IGFBP-3 and IGFBP-4 [101]. Bone mineral density (BMD) at the radius midshaft showed a positive correlation with serum IGF-I and a negative correlation with IGFBP-4. From the results, we proposed that thyroid hormone stimulated effects to increase inhibitory IGF-I binding proteins could modulate effects of thyroid hormone in hyperthyroid patients and potentially contribute to the observed bone loss [101]. We also pursued the other side of thyroid hormone action, the effects of thyroid hormone to stimulate resorption. In earlier in vitro studies by Greg Mundy and Larry Raisz, thyroid hormone appeared to have a lower efficacy (maximal response) than PTH in stimulating resorption in the bone organ cultures [102]. We investigated the role of paracrine effects, through intermediate factors, in the resorptive response. Oskar Hoffmann, then a student with Meinrad Peterlik in Vienna, had found that the effects of thyroxin on calvarial resorption were prostaglandindependent [103]. Oskar came to my laboratory to determine if there were similar effects on limb bones. He found that the resorptive effect in limb bones was independent of prostaglandins [104]. We then examined the role of cytokines in thyroid hormone action. Gabor Tarjan showed that thyroid hormones potentiated the effects of IL-1 to increase IL-6 production by osteoblastic cells and in bone organ cultures and also promoted the in vitro bone resorption elicited by IL-1 [105]. In a collaboration with Peter Lakatos and his colleagues in Hungary, Gabor showed correlations of IL-6 and bone metabolism in patients with thyroid function disorders [106]. The role of growth factors and cytokines in thyroid hormone action is still a topic of interest, as other studies have reported that thyroid hormones alone do not have significant effects on RANKL [107,108], although co-treatment with calcitriol promoted thyroid hormone effects on RANKL expression [109]. The

interaction of thyroid hormone with other factors is apparent from literature showing that low estrogen can augment thyroid hormone-induced bone loss [110,111]. ENDOTHELIN

Interest in endothelins (ET) initially derived from their vascular effects [112]. Because ET-I had been shown to affect calcium transients in osteoblastic cells [113], and, as will be discussed later, we were interested in calcium signaling in bone, we decided to investigate mechanisms of this effect as well as their actions on bone formation and resorption. David Semler, a Ph.D. student, identified ETA and ETB receptors in osteoblastic cells and showed that they were both functional in signaling. He also found that eight-hour preculture with PTH downregulated ET-stimulated calcium signaling [114]. In other calcium signaling studies, Suk Kyeong Lee, a Ph.D. student, made the interesting observation that brief pretreatment with ET or selective ETB receptor activators markedly increased PTH-stimulated calcium signaling through a mechanism that remains to be elucidated [115]. Agnes Tatrai, a research associate and Peter Lakatos, together with others in the laboratory, found that ET-I did not affect cyclic adenosine monophosphate (cAMP), but increased phospholipase A2, C, and D (PLD) activity in bone cells [116–118]. Consistent with the effect on phospholipase A2, ET stimulated prostaglandin-dependent resorption; however, it also stimulated collagen and noncollagenous protein synthesis, effects that were prostaglandin independent [117]. Peter’s colleagues in Hungary obtained serum from patients, and in a collaborative study, we found that that patients with primary and secondary hyperparathyroidism had elevated serum ET levels [119]. Consistent with this, PTH also stimulated ET-1 production by fetal rat limb bones [118]. These observations suggest that PTH–ET interactions may be important in normal bone physiology. DIURNAL RHYTHMS IN BONE REMODELING

Around the same time when trainees and collaborators from Austria and Hungary were joining the laboratory, I began exciting and fruitful collaborations with investigators from Japan. The first Japanese collaborator was Prof. Hisashi Shinoda, who came as a visiting scientist first from Tokyo and then from Tohoku University in Sendai. One of Hisashi’s areas of expertise was diurnal rhythms in bone, and he thought that the bone organ culture model would be a good system for examining this. In another of those heroic, labor-intensive series of experiments, Hisashi would spend the night in the laboratory in order to bleed 45Ca-prelabeled rats, maintained on a dark/light cycle, every four hours to determine calcium deposition into bone and calcium release. He also determined the bone-resorbing activity of the serum in the bone organ cultures. He found peak

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bone resorbing activity in serum obtained at 8 A.M., with a later peak in serum calcium [120] (Fig. 5.11). We extended the culture studies to human samples, in a collaboration between our laboratory and Richard Eastell and Aubrey Blumsohn in Sheffield (UK). The findings showed a circadian rhythm in vitro bone-resorbing activity in human serum as well, with a peak at around 1 A.M. [121] (Fig. 5.11). To attempt to assess what the factor might be, we used a neutralizing antibody to human PTH (a gift from Jack Martin) and the cortisol antagonist, RU 486. The antibody did not alter the pattern; however, RU 486 prevented the trough in resorptive activity (Fig. 5.11). The timing of the antiresorptive activity correlated with the peak in cortisol concentrations, and was

consistent with our earlier findings of inhibitory effects of cortisol on bone resorption in the organ cultures [50]. SIGNALING IN BONE CELLS: EXPLORING A NOVEL OSTEOBLAST SIGNALING PATHWAY WITH A GREAT TEAM OF STUDENTS AND FELLOWS  A major thread

of research activity of the laboratory that started as far back as the 1970s but became more prominent in recent years has been the investigation of cAMP-independent signaling in osteoblasts. We did not intentionally set out to avoid cAMP, but it followed from experiments carried out by Rosemary Dziak. Rosemary, in her Ph.D. work with John Brand, had shown that cells isolated from neonatal mouse calvaria responded to PTH with a Calcium Transfer into Bone Calcium Release from Bone Bone Resorbing Activity of Serum

% Bone 45Ca Released

25% Serum 25% Serum • 0.5 µm Indomethacin 60 50

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Sampling Time (hours)

Sampling Time (hours)

FIGURE 5.11  Upper left: diurnal rhythm of bone-resorbing activity of rat serum collected at different times of day. Serum was collected at times indicated on the abscissa from male rats adapted to a 12:12 light:dark cycle. Fetal rat ulnae were incubated in media prepared by supplementing Dulbecco's Modified Eagle's medium (DMEM) with 25% serum from indicated time points. Values are means ± standard error of the mean (SEM). *, # p < 0.05, ## p < 0.01 versus peak value at 0800 A.M. Upper right: chronological relationship among calcium transfer into bone, calcium release from bone and bone-resorbing activity of serum collected at different times of day. Source: Shinoda and Stern (1992) [120]. Lower left: diurnal rhythm of bone-resorbing activity in sera (SBRA) from women and men. Values are means ± SEM. Lower right: the glucocorticoid antagonist RU 486 (1 μM) (line with stars) suppresses the rhythm of SBRA. Source: Lakatos et al. (1995) [121].

I. INTRODUCTION

20

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5.  OSTEO-ODYSSEY: A MEMOIR

% Decrease Bone Calcium

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FIGURE 5.12  Calcium ionophore A23187 stimulates resorption of fetal rat bone organ cultures. Upper: dose-dependent effects of 72 culture with A23187 on 45Ca release, % collagen and calcium. Source: with kind permission from Springer Science and Business Media B.V. [124]. Lower: numerous osteoclasts are seen in fetal rat limb bone cultured for 48 hours with 6 × 10–7 M A23187. Source: Stern et al. (1982) [123].

rapid increase in calcium uptake. Our follow-up studies in my laboratory revealed that this PTH effect was independent of cAMP, and that the calcium ionophore, A23187, had a biphasic effect to promote osteoclastogenesis and bone resorption [122–124] (Fig. 5.12). Our publications on ionophores led to an invitation from Marshall Urist to write a review on the topic for Clinical Orthopedics [125]. This was a comprehensive piece of work; however, I doubt that many people read it. Evidence that this is the case was the lack of comment on a sentence that I had included in the section describing effects of ionophores on development of the ovum. Ionophores elicited several division steps, and in my draft, I included the sentence, “Longer term experiments will determine whether ionophores can be a replacement for men.” The editorial review either missed this or enjoyed the humor and left it in, and it ended up in the final document. Only two people commented on it to me. One was an MD/PhD student who was doing a rotation in my laboratory. I was surprised that he had read the review. The other person

was Larry Raisz, who called and said, “I can’t believe you got away with that!” The findings on calcium led to studies to investigate effects of PTH, thrombin, and ET on phospholipase C and phosphatidylinositol metabolism in osteoblasts and bone, carried out by Mark Rappaport, Geetha Shankar, Agnes Tatrai, and Peter Lakatos [126–129]. As we began the studies, I realized that I needed to get a better feel for phospholipid signaling by working with experts, and spent two summers on sabbatical in Dennis Vance’s laboratory at the University of British Columbia (UBC). Sabbaticals can have multiple positive consequences. In addition to making me more knowledgeable on the topic and resulting in a paper with Dennis on phosphatidylcholine metabolism in bone [130], I met a lipid researcher from Japan, who in turn introduced me to Prof. Shinoda (see “Diurnal Rhythms In Bone Remodeling,’’ above). Also at UBC, I met Harold Copp, and was able to put together a manuscript on stanniocalcin that included studies from him, Nell Hermann-Erlee, David Goltzman, and Kanji Sato [131]. An additional consequence of the sabbatical was that my husband and I became enamored with British Columbia and have returned there for vacation every year since. Exploring downstream consequences of phospholipase C activation led to studies by Pam Stewart and Vicky Stathapoulos revealing effects of PTH on c­ alcium/ phosphatidylserine-dependent phosphorylation [132], and on diacyglycerol production in response to PTH 1-34 and PTH 3-34 amide [133]. These findings led us to focus on protein kinase C (PKC), especially the isozymes dependent upon calcium and diacylglycerol. The PKC work, carried out by PhD students, Jennifer Sanders, Debra Dossing, and Julie Radeff, and Zsolt Nagy, a third physician-scientist fellow from Semmelweis University, has been particularly exciting, as it moved us in an unanticipated direction, toward recognizing a new signaling pathway and thinking about interactions between G protein signaling pathways. The studies identified which PKC isoforms were present in a number of murine, rodent, and human cell lines and normal human cells [134], and demonstrated that several of these were translocated by PTH (Fig. 5.13), IL-1β, and TNF-α [135,136]. The PTH-stimulated translocation of the PKC-α and -β isoforms was independent of cAMP [137] (Fig. 5.13). The work took an unexpected turn away from phospholipase C-mediated signaling when we found that the phospholipase C inhibitor, U-73122, had only minimal effects on PTH-mediated PKC translocation, and furthermore, that the effects of U-73122 were replicated by an inactive analog [137]. This and other evidence suggested the possibility that the PTHstimulated PKC generation was mediated, not through phospholipase C, but possibly through PLD, which generates diacylglycerol indirectly through the production of phosphatidic acid, which is then converted

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Cytosol

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FIGURE 5.13  Upper: parathyroid hormone (PTH) (1 nM, 1 min) elicits membrane translocation of protein kinase Cα (PKCα) and PKCβ in UMR-106 osteoblastic cells as detected by Western blotting. Subcellular fractions were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted with PKC isozyme specific antibodies. Detection was carried out by chemiluminescence and quantified by densitometry. Values are % change in PTH-treated cultures compared with control cultures. Source: Dossing et al. (2001) [135]. Middle: the PKCα translocation is elicited by both PTH 1-34 and PTH 3-34 amide and is independent of protein kinase A (PKA) (not elicited by FSK and not inhibited by the PKA antagonist PKI). Source: Radeff et al. (2004) [137] and J. Radeff, Ph.D. thesis. Cells were grown on coverslips and treated for 5 min. Cells were fixed and permeabilized, and isozymes detected using PKC-specific antibodies and FITC-conjugated secondary antibodies. Confocal microscopy was used to visualize the localization of the isozymes. Lower: the PTH-stimulated translocation is blocked by transient expression of dominant negative Rho A (Rho A 19 N) and mimicked by constitutively active Rho A (Rho A 63L). Source: Radeff et al. (2004) [140].

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TRAP Activity (Absorbance 410nm)

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although the osteoclastogenesis stimulated by RANKL was unaffected [144]. Consistent with these effects on osteoclastogenesis, the effect of PTH plus calcitriol to increase RANKL was prevented in cells expressing the constitutively active constructs (Fig. 5.14). The results suggested a potential feedback regulatory role of G12/ Rho A-mediated signaling on hormone-stimulated osteoclastogenesis. Rho A signaling also had effects on the osteoblasts independent of PTH. Tomohiko Yoshida, an endocrinologist from Chiba, found that Rho A mimicked the effect of PTH to promote osteoblast survival, but was not required for the PTH effect [145]. Rho A signaling was also important for actin cytoskeletal integrity of the osteoblasts. A medical student, Nikolas Kazmers, found that constitutively active RhoA antagonized the cytoskeletal breakdown elicited by the bisphosphonate alendronate or by a geranylgeranyl transferase inhibitor, but not that elicited by a downstream Rho kinase inhibitor [146] (Fig. 5.15). This effect of Rho A signaling

ol

to diacyglycerol by a phosphatidic acid phosphatase. Studies on PLD carried out by a faculty research associate, Amareshwar Singh, indicated that PTH can activate PLD signaling [138], through Gα12 and Gα13 heterotrimeric G protein family members [139]. G12/13 signaling transduction is frequently mediated through guanine nucleotide exchange factors affecting Rho family small G proteins, and our studies showed that Rho A (Fig. 5.13) and the downstream kinase Rho kinase regulate PTH-stimulated PKC-α translocation [140] in addition to being involved with PLD activation [139,141]. We had found earlier that PKC contributes to the IL-6 expression elicited by PTH [142,143], and further studies showed that Rho A and Rho kinase contribute to PTH-stimulated IL-6 production [140], and are involved in PTH effects on PLD [141]. A particularly intriguing finding was that osteoblasts expressing constitutively active Rho A or Gα12 had markedly attenuated PTH plus calcitriolstimulated osteoclastogenesis in co-cultures (Fig. 5.14),

RANKL

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FIGURE 5.14  Upper left: stable expression of constitutively active Gα12 or constitutively active Rho A in UMR-106 cells inhibits parathyroid hormone (PTH) + calcitriol-stimulated osteoclastogenesis in co-cultures with RAW 264.7 osteoclast precursor cells. Upper right: the constitutively active constructs did not inhibit the direct effect of RANKL on osteoclastogenesis in the RAW 264.7 cells. Lower: stable expression of constitutively active Gα12 or constitutively active Rho A inhibit PTH + calcitriol-stimulated RANKL expression and prevent PTH + calcitriol inhibition of osteoprotegerin expression in UMR-106 cell cultures. Source: Wang and Stern (2010) [144].

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to protect the actin cytoskeleton was mimicked by PTH 3-34 amide, but not by PTH 1-34 [146], which was not surprising, since PTH 1-34 is known to promote breakdown of the actin cytoskeleton through increased cAMP [147,148]. The findings on the actions of Gα12 led us to think about interactions between G protein signaling pathways in PTH action. In work on PTH signaling carried out by Jun Wang, a research faculty member, we explored the contributions of Gαs, Gαq, and Gα12 pathways in PTH actions on gene expression in osteoblastic cells using a small (169 gene) osteogenesis and signaling gene array [149]. To determine the contributions of each of the G proteins, we used mini-genes encoding antagonists of each of the G proteins, which we had also used in the studies of the G protein regulation of PLD [139]. Both of the studies were carried out in collaboration with Annette Gilchrist, who developed the mini-genes. We found that PTH regulated 32 genes in the array. Of these, some genes regulated exclusively by only one G protein, some were regulated by two, some were regulated by all three, and two genes were not regulated by any of the three G proteins (Fig. 5.16). Some differences in time

(A)

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FIGURE 5.16  Contributions of Gαs, Gαq, and Gα12 to parathyroid hormone (PTH) effects on 32 target genes, as shown by inhibition by minigenes encoding inhibitors to these three G proteins. The study revealed examples of overlapping regulation as well as a few genes that were unaffected by the minigenes. Source: Wang et al. (2011) [149].

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FIGURE 5.15  Constitutively active Rho A (Rho A 63L) overcomes the disrupting effects of alendronate and the geranylgeranyl transferase inhibitor GGTI-2166, but not of the downstream Rho kinase inhibitor Y27632 on the integrity of the actin cytoskeleton of MC3T3-E1 cells. Source: Kazmers et al. (2009) [146].

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course were also detected, suggesting that in addition to dual or triple control, there could be sequential activation of pathways. The results were exciting and hopefully will be extended in a larger screen. The intriguing new concepts that gene array approaches can reveal showed up in two other of our studies. In one, we observed dose and time differences in gene expression after treatment of osteoblastic cells with the bisphosphonate risedronate, with effects on growth factors being prominent with low doses and short exposure [150]. In the other study, we found marked gender differences in responses of human cells from male versus female donors to both estrogen and testosterone [151]. I hope to pursue both of these findings in future studies. I have titled this memoir as an odyssey, perhaps inspired by the similarity of the pattern of the resorbed calvaria to the map of the travels of Odysseus (Fig. 5.17). A lifetime of research is indeed an odyssey, with techniques being the craft carrying the work, findings in the field being the winds that drive the work into interesting harbors, and colleagues as the shipmates. There have been other projects, and I have not been able to mention every one of the approximately 100 mentees, from high-school students to junior faculty who has worked with me and provided fascinating and thought-provoking findings. People with interests and expertise outside of mine brought interesting work to the laboratory, such as Joseph Kunnel, now an orthodontist, who as a PhD student developed a novel device for studying loading and the associated signaling [152,153], Tomohiko Yoshida, who in addition to his work on Rho signaling, developed a proposal and carried out studies showing both direct inhibitory and indirect stimulatory effects of the RAGE (receptor for advanced glycation end products) ligand S-100 on

osteoclastogenesis [154] and Jae Woo, who brought fascinating compounds such as compactin [155] and reveromycin [156] to the laboratory for studies on resorption and osteoclastogenesis. We had stimulating collaborations with Marika Bhattacharyya in her work on cadmium and bone loss [157], with Lynn Matrisian and metalloproteinases correlating with resorption [158], and with Buck Strewler and Bob Nissenson on transforming growth factor-α [159] and “parathyroid hormonelike protein” [160]. I am grateful to have had two dedicated long-term technicians, Thalia Mavreas and Shirley Foster, who contributed in many ways to our research.

THE BONE WORLD BEYOND THE LABORATORY: ASBMR AND NOF My involvement in activities outside the laboratory in the world of bone policy probably arose from several factors. From the start of my faculty appointment, I began to participate in policy activities at my own institution, such as faculty governance. Larry Raisz might have been a role model even for this aspect of my professional career. His participation in study sections and professional societies made it seem an important responsibility for a scientist. As mentioned previously, in the early 1970s it was recognized that women were underrepresented on critical review panels. The timing was such that early on in my career, I was asked to serve on review committees, such as Food and Drug Administration (FDA) panels, NASA review panels, and once my NIH grant had been renewed after the first three-year funding period, on study sections. I served two three-year terms on the General Medicine B Study Section. When the American Society for Bone and

FIGURE 5.17  Similarities in the pattern of resorption in neonatal mouse calvaria [10] and the map of Odysseus' travels (figure from Wikipedia).

I. INTRODUCTION

The Bone World Beyond the Laboratory: ASBMR and NOF

Mineral Research (ASBMR) formed in the late 1970s, I became a member, and served on the first council. In 1984, I became the first female president of the society. Relevant to this memoir being in the Osteoporosis book is that at the time I was President-elect of ASBMR, a major priority of the society, initiated the year before my presidency by Bill Peck, was the formation of an osteoporosis foundation. I still have the letter that was sent to a group of leaders in the field inviting them to a luncheon at the 1984 ASBMR meeting at which we discussed 1) whether there was general support for such an endeavor, 2) what the goals should be, 3) the nature of the steering committee, 4) the costs of establishing the organization, 5) other model foundations, and 6) how the functions of the foundation would relate to other public education activities of the ASBMR. It must have been a long lunch. The following year, when I was President of ASBMR, the Osteoporosis Foundation, later to be renamed the National Osteoporosis Foundation (NOF), was founded by ASBMR. Bill Peck was the first NOF President, and Larry Riggs and I were the first Vice-Presidents. The other major activity during the year of my presidency of ASBMR was laying the groundwork for the establishment of the Journal of Bone and Mineral Research, which became a reality the following year, the year of Larry Riggs’ presidency of ASBMR. The research presented in the journal and at the meetings reflects the breadth and depth of bone research. ASBMR has, from the outset, been an organization that is supportive of both men and women scientists. The fact that a number of outstanding women have served as ASBMR President in recent years reflects well on both the bone community and on those who have been willing to serve. The organization also has had a continuing strong commitment to young scientists, and has always provided them with the opportunity to present their research and become recognized. It is also exciting and important that the international bone community is a large and strong component of ASBMR, helping to make for a seamless bone world. I am delighted to have had the opportunity to have been involved with both ASBMR and NOF from their outsets and to have been invited to be continually involved in science policy activities facilitating the future of bone research. Finally, I thank the editors for the invitation to reflect back and write this memoir.

References [1]  Au WY, Poland AP, Stern PH, Raisz LG. Hormone synthesis and secretion by rat parathyroid glands in tissue culture. J Clin Invest 1970;49(9):1639–46. [2]  Stern PH, Raisz LG. An analysis of the role of serum in parathyroid hormone-induced bone resorption in tissue culture. Exp Cell Res 1967;46(1):106–20. [3]  Stern PH, Miller JC, Chen SF, Kahn DJ. A bone resorbing substance from bovine serum albumin (brA). Calcif Tissue Res 1978;25(3):233–40.

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[4]  Stern PH. Albumin-induced resorption of fetal rat bone in vitro. Calcif Tissue Res 1971;7(1):67–75. [5]  Hefley TJ, Stern PH. Isolation of osteoclasts from fetal rat long bones. Calcif Tissue Int 1982;34(5):480–7. [6]  Parkhill JE, Tarjan G, Chai JY, Stern PH. An in vitro model for studying oral bone loss. Northwest Dent Res 1996;6(2):17–20. [7]  Stewart PJ, Stern PH. Vertebral bone resorption in vitro: effects of parathyroid hormone, calcitonin, 1,25 dihydroxyvitamin D3, epidermal growth factor, prostaglandin E2, and estrogen. Calcif Tissue Int 1987;40(1):21–6. [8]  Stern P, Krieger N. Comparison of fetal rat limb bones and neonatal mouse calvaria: Effects of parathyroid hormone and 1,25-dihydroxyvitamin D3. Calcif Tissue Int 1983;35:172–6. [9]  Hefley TJ, Krieger NS, Stern PH. Simultaneous measurement of bone resorption and collagen synthesis in neonatal mouse calvaria. Anal Biochem 1986;153(1):166–71. [10] Stock SR, Ignatiev KI, Foster SA, Forman LA, Stern PH. MicroCT quantification of in vitro bone resorption of neonatal murine ­calvaria exposed to IL-1 or PTH. J Struct Biol 2004;147(2):185–99. [11]  Bell NH, Stern PH. Effects of changes in serum calcium on ­hypocalcemic response to thyrocalcitonin in the rat. Am J Physiol 1970;218(1):64–8. [12] Barrett RJ, Bell NH, Stern PH, Schlueter RJ. Extraction, partial purification and demonstration of biological activity of human thyrocalcitonin. J Clin Endocrinol Metab 1968;28(5):734–9. [13] Stern PH, Bell NH. Effects of glucagon on serum calcium in the rat and on bone resorption in tissue culture. Endocrinology 1970;87(1):111–7. [14] Gozariu L, Minne H, Ziegler R. Rat bone osteolysis in vitro: ­inhibitory effects of glucagon and cortisol. Horm Metab Res 1971;3(3):225–6. [15] Woodward AH, Jowsey J. The effects of glucagon on immobilization osteoporosis in rats. Endocrinology 1972;90(5):1399–401. [16] Kalu DN, Hillyard C, Foster GV. Effect of glucagon on bone collagen metabolism in the rat. J Endocrinol 1972;55(2):245–52. [17] Yamada C, Yamada Y, Tsukiyama K, Yamada K, Udagawa N, Takahashi N, et al. The murine glucagon-like peptide-1 receptor is essential for control of bone resorption. Endocrinology 2008;149(2):574–9. [18] Clowes JA, Khosla S, Eastell R. Potential role of pancreatic and enteric hormones in regulating bone turnover. J Bone Miner Res 2005;20(9):1497–506. [19] Gottschalck IB, Jeppesen PB, Hartmann B, Holst JJ, Henriksen DB. Effects of treatment with glucagon-like peptide-2 on bone resorption in colectomized patients with distal ileostomy or jejunostomy and short-bowel syndrome. Scand J Gastroenterol 2008;43(11):1304–10. [20] Stern PH, Hamstra AJ, DeLuca HF, Bell NH. A bioassay capable of measuring 1 picogram of 1,25-dihydroxyvitamin D3. J Clin ­Endocrinol Metab 1978;46(6):891–6. [21] Stern PH, Phillips TE, Mavreas T. Bioassay of 1,25-dihydroxyvitamin D in human plasma purified by partition, alkaline extraction and high-pressure chromatography. Anal Biochem 1980;102:22–30. [22] Brooks MH, Bell NH, Love L, Stern PH, Orfei E, Queener SF, et al. Vitamin-D-dependent rickets type II. Resistance of target organs to 1,25-dihydroxyvitamin D. N Engl J Med 1978;298(18):996–9. [23] Bell NH, Epstein S, Stern PH. Hypercalcemia during long-term treatment with 1,25-dihydroxyvitamin D3 in hypoparathyroidism. N Engl J Med 1979;301(21):1183–4. [24] Bell NH, Stern PH. Hypercalcemia and increases in serum hormone value during prolonged administration of 1alpha,25-dihydroxyvitamin D. N Engl J Med 1978;298(22):1241–3. [25] Stern PH, De Olazabal J, Bell NH. Evidence for abnormal regulation of circulating 1 alpha,25-dihydroxyvitamin D in patients with sarcoidosis and normal calcium metabolism. J Clin Invest 1980;66(4):852–5.

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[26] Bell NH, Stern PH, Pantzer E, Sinha TK, DeLuca HF. Evidence that increased circulating 1 alpha, 25-dihydroxyvitamin D is the probable cause for abnormal calcium metabolism in sarcoidosis. J Clin Invest 1979;64(1):218–25. [27] Epstein S, Stern PH, Bell NH, Dowdeswell I, Turner RT. Evidence for abnormal regulation of circulating 1 alpha, 25-dihydroxyvitamin D in patients with pulmonary tuberculosis and normal calcium ­metabolism. Calcif Tissue Int 1984;36(5):541–4. [28] Lambert PW, Stern PH, Avioli RC, Brackett NC, Turner RT, Greene A, et al. Evidence for extrarenal production of 1 alpha,25dihydroxyvitamin D in man. J Clin Invest 1982;69(3):722–5. [29] Bell NH, Stern PH, Paulson SK. Tight regulation of circulating 1 alpha,25-dihydroxyvitamin D in black children. N Engl J Med 1985;313(22):1418. [30] Stern PH, Taylor AB, Bell NH, Epstein S. Demonstration that ­circulating 1 alpha, 25-dihydroxyvitamin D is loosely regulated in normal children. J Clin Invest 1981;68(5):1374–7. [31] Paulson SK, Perlman K, DeLuca HF, Stern PH. 24- and 26-homo-­ 1,25-dihydroxyvitamin D3 analogs: potencies on in vitro bone ­resorption differ from those reported for cell differentiation. J Bone Miner Res 1990;5(2):201–6. [32] DeLuca HF, Sicinski RR, Tanaka Y, Stern PH, Smith CM. Biological activity of 1,25-dihydroxyvitamin D2 and 24-epi-1,25-dihydroxyvitamin D2. Am J Physiol 1988;254(4 Pt 1):E402–6. [33] Stern PH, Mavreas T, Tanaka Y, DeLuca HF, Ikekawa N, Kobayashi Y. Fluoride substitution of vitamin D analogs at C-26 and C-27: enhancement of activity of 25-hydroxyvitamin D but not of 1,25-dihydroxyvitamin D on bone and intestine in vitro. J Pharmacol Exp Ther 1984;229(1):9–13. [34] Stern PH, Halloran BP, DeLuca HF, Hefley TJ. Responsiveness of vitamin D-deficient fetal rat limb bones to parathyroid hormone in culture. Am J Physiol 1983;244(4):E421–4. [35] Stern PH, Tanaka Y, DeLuca HF, Ikekawa N, Kobayashi Y. Bone resorptive activity of side-chain fluoro derivatives of 25-hydroxy- and 1 alpha,25-dihydroxyvitamin D3 in culture. Mol Pharmacol 1981;20(3):460–2. [36] Napoli JL, Fivizzani MA, Hamstra AH, Schnoes HK, DeLuca HF, Stern PH. The synthesis and activity in vitro of 25-masked 1alphahydroxylated vitamin D3 analogs. Steroids 1978;32(4):453–66. [37] Stern PH, Ness EM, DeLuca HF. Responses of fetal rat bones to Solanum malacoxylon in vitro: a possible explanation of previous paradoxical results. Mol Pharmacol 1978;14(2):357–65. [38] Stern PH, Trummel CL, Schnoes HK, Deluca HF. Bone resorbing activity of vitamin D metabolites and congeners in vitro: influence of hydroxyl substituents in the A ring. Endocrinology 1975;97(6):1552–8. [39] Stern PH, DeLuca HF, Ikekawa N. Bone resorbing activities of 24-hydroxy stereoisomers of 24-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. Biochem Biophys Res Commun 1975;67(3):965–71. [40] Lam HY, Schnoes HK, DeLuca HF, Reeve L, Stern PH. Structural analogs of 1alpha,25-dihydroxycholecalciferol: preparation and biological assay of 1alpha-hydroxypregnacalciferol. Steroids 1975;26(4):422–36. [41] Stern PH, Rappaport MS, Mayer E, Norman AW. 24-Oxo and 26,23-lactone metabolites of 1,25-dihydroxyvitamin D3 have direct bone-resorbing activity. Arch Biochem Biophys 1984;230(2):424–9. [42] Stern PH, Horst RL, Gardner R, Napoli JL. 10-Keto or 25-hydroxy substitution confer equivalent in vitro bone-resorbing activity to vitamin D3. Arch Biochem Biophys 1985;236(2):555–8. [43] Stern PH. A monolog on analogs: in vitro effects of vitamin D ­metabolites and consideration of the mineralization question. Calcif Tissue Int 1981;33(1):1–4. [44] Jones G. Vitamin D analogs. Endocrinol Metab Clin North Am 2010;39(2):447–72. [45] Stern PH. The D vitamins and bone. Pharmacol Rev 1980;32(1): 47–80.

[46] Gelbard HA, Stern PH, U’Prichard DC. 1 alpha, 25-Dihydroxyvitamin D3 nuclear receptors in pituitary. Science 1980;209(4462):1247–9. [47] Gelbard HA, Stern PH, U’Prichard DC. Characteristics of [3H]1 alpha, 25-(OH)2D3 binding to nuclear fractions from rat pituitary adenoma GH3 cells. Life Sci 1981;29(10):1051–6. [48] Sachs RH, Gelbard HA, U’Prichard DC, Stern PH. Specific 3H-­ calcitriol binding in bovine brain and transformed neural cell lines. Fed Proc 1982;41:1708. [49] Dossing DA, Stern PH. Receptor activator of NF-kappaB ligand protein expression in UMR-106 cells is differentially regulated by parathyroid hormone and calcitriol. J Cell Biochem 2005; 95(5):1029–41. [50] Stern PH. Inhibition by steroids of parathyroid hormone-­ induced Ca45 release from embryonic rat bone in vitro. J Pharmacol Exp Ther 1969;168(2):211–7. [51] Feldman D, Dziak R, Koehler R, Stern P. Cytoplasmic glucocorticoid binding proteins in bone cells. Endocrinology 1975;96(1):29–36. [52] Choe J, Stern P, Feldman D. Receptor mediated glucocorticoid inhibition of protein synthesis in isolated bone cells. J Steroid ­Biochem 1978;9(3):265–71. [53] Peck WA, Brandt J, Miller I. Hydrocortisone-induced inhibition of protein synthesis and uridine incorporation in isolated bone cells in vitro. Proc Natl Acad Sci U S A 1967;57(6):1599–606. [54] Delany AM, Gabbitas BY, Canalis E. Cortisol downregulates osteoblast alpha 1 (I) procollagen mRNA by transcriptional and posttranscriptional mechanisms. J Cell Biochem 1995;57(3):488–94. [55] Yao W, Cheng Z, Busse C, Pham A, Nakamura MC, Lane NE. Glucocorticoid excess in mice results in early activation of osteoclastogenesis and adipogenesis and prolonged suppression of osteogenesis: a longitudinal study of gene expression in bone tissue from glucocorticoid-treated mice. Arthritis Rheum 2008; 58(6):1674–86. [56] Weinstein RS, Jilka RL, Parfitt AM, Manolagas SC. Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their ­deleterious effects on bone. J Clin Invest 1998;102(2):274–82. [57] Hofbauer LC, Gori F, Riggs BL, Lacey DL, Dunstan CR, Spelsberg TC, et al. Stimulation of osteoprotegerin ligand and inhibition of osteoprotegerin production by glucocorticoids in human osteo­blastic lineage cells: potential paracrine mechanisms of g ­lucocorticoidinduced osteoporosis. Endocrinology 1999;140(10):4382–9. [58] Shin HH, Kim SJ, Kang SY, Lee DS, Choi HS. Soluble glucocorticoid-induced tumor necrosis factor receptor stimulates osteoclastogenesis by down-regulation of osteoprotegerin in bone marrow stromal cells. Bone 2006;39(4):716–23. [59] Dovio A, Perazzolo L, Saba L, Termine A, Capobianco M, Bertolotto A, et al. High-dose glucocorticoids increase serum levels of soluble IL-6 receptor alpha and its ratio to soluble gp130: an additional mechanism for early increased bone resorption. Eur J Endocrinol 2006;154(5):745–51. [60] Kim YH, Jun JH, Woo KM, Ryoo HM, Kim GS, Baek JH. Dexamethasone inhibits the formation of multinucleated osteoclasts via down-regulation of beta3 integrin expression. Arch Pharm Res 2006;29(8):691–8. [61] Jia D, O’Brien CA, Stewart SA, Manolagas SC, Weinstein RS. Glucocorticoids act directly on osteoclasts to increase their life span and reduce bone density. Endocrinology 2006;147(12):5592–9. [62] Sivagurunathan S, Muir MM, Brennan TC, Seale JP, Mason RS. Influence of glucocorticoids on human osteoclast generation and activity. J Bone Miner Res 2005;20(3):390–8. [63] Stewart PJ, Green OC, Stern PH. Possible cyclosporine induced pseudohypoparathyroidism: clinical observations and inhibition of bone resorption in vitro by cyclosporine. Clin Res 1985;33(4):A905. [64] Stewart PJ, Green OC, Stern PH. Cyclosporine A inhibits calcemic hormone-induced bone resorption in vitro. J Bone Miner Res 1986;1(3):285–91.

I. INTRODUCTION

The Bone World Beyond the Laboratory: ASBMR and NOF

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5.  OSTEO-ODYSSEY: A MEMOIR

[105] Tarjan G, Stern PH. Triiodothyronine potentiates the stimulatory effects of interleukin-1 beta on bone resorption and medium interleukin-6 content in fetal rat limb bone cultures. J Bone Miner Res 1995;10(9):1321–6. [106] Lakatos P, Foldes J, Horvath C, Kiss L, Tatrai A, Takacs I, et al. Serum interleukin-6 and bone metabolism in patients with thyroid function disorders. J Clin Endocrinol Metab 1997;82(1):78–81. [107] Saraiva PP, Teixeira SS, Padovani CR, Nogueira CR. Triiodothyronine (T3) does not induce Rankl expression in rat Ros 17/2.8 cells. Arq Bras Endocrinol Metabol 2008;52(1):109–13. [108] Kanatani M, Sugimoto T, Sowa H, Kobayashi T, Kanzawa M, Chihara K. Thyroid hormone stimulates osteoclast differentiation by a mechanism independent of RANKL-RANK interaction. J Cell Physiol 2004;201(1):17–25. [109] Miura M, Tanaka K, Komatsu Y, Suda M, Yasoda A, Sakuma Y, et al. A novel interaction between thyroid hormones and 1,25(OH)(2)D(3) in osteoclast formation. Biochem Biophys Res Commun 2002;291(4):987–94. [110] Zeni S, Gomez-Acotto C, Di Gregorio S, Mautalen C. Differences in bone turnover and skeletal response to thyroid hormone treatment between estrogen-depleted and repleted rats. Calcif Tissue Int 2000;67(2):173–7. [111] Faber J, Galloe AM. Changes in bone mass during prolonged subclinical hyperthyroidism due to L-thyroxine treatment: a meta-analysis. Eur J Endocrinol 1994;130(4):350–6. [112] Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332(6163):411–5. [113] Takuwa Y, Ohue Y, Takuwa N, Yamashita K. Endothelin-1 activates phospholipase C and mobilizes Ca2+ from extra- and intracellular pools in osteoblastic cells. Am J Physiol 1989;257(6 Pt 1):E797–803. [114] Semler DE, Morris DL, Stern PH. Endothelin-stimulated Ca(2+) signaling and endothelin receptor expression are decreased by parathyroid hormone treatment in UMR-106 osteoblastic osteosarcoma cells. Cell Calcium 2000;28(1):55–64. [115] Lee SK, Stern PH. EndothelinB receptor activation enhances parathyroid hormone-induced calcium signals in UMR-106 cells. J Bone Miner Res 1995;10(9):1343–51. [116] Tatrai A, Lakatos P, Thompson S, Stern PH. Effects of endothelin-1 on signal transduction in UMR-106 osteoblastic cells. J Bone Miner Res 1992;7(10):1201–9. [117] Tatrai A, Foster S, Lakatos P, Shankar G, Stern PH. Endothelin-1 actions on resorption, collagen and noncollagen protein synthesis, and phosphatidylinositol turnover in bone organ cultures. Endocrinology 1992;131(2):603–7. [118] Stern PH, Tatrai A, Semler DE, Lee SK, Lakatos P, Strieleman PJ, et al. Endothelin receptors, second messengers, and actions in bone. J Nutr 1995;125(7 Suppl):2028S–32S. [119] Lakatos P, Tatrai A, Foldes J, Horvath C, Mako J, Stern PH. Endothelin concentrations are elevated in plasma of patients with primary and secondary hyperparathyroidism. Calcif Tissue Int 1996;58(1):70–1. [120] Shinoda H, Stern PH. Diurnal rhythms in Ca transfer into bone, Ca release from bone, and bone resorbing activity in serum of rats. Am J Physiol 1992;262(2 Pt 2):R235–40. [121] Lakatos P, Blumsohn A, Eastell R, Tarjan G, Shinoda H, Stern PH. Circadian rhythm of in vitro bone-resorbing activity in ­human ­serum. J Clin Endocrinol Metab 1995;80(11):3185–90. [122] Dziak R, Stern PH. Calcium transport in isolated bone cells. III. Effects of parathyroid hormone and cyclic 3’,5’-AMP. Endocrinology 1975;97(5):1281–7. [123] Stern PH, Orr MF, Brull E. Ionophore A23187 promotes osteoclast formation in bone organ culture. Calcif Tissue Int 1982;34(1):31–6.

[124] Stern PH. Interactions of calcemic hormones and divalent cation ionophores on fetal rat bone in vitro. In: Rubin RP, Weiss GB, Putney Jr JW, editors. Calcium in Biological Systems. New York: Plenum; 1985. p. 541–7. [125] Stern PH. Ionophores: chemistry, physiology and potential applications to bone biology. Clin Orthop Relat Res 1977;122:273–98. [126] Rappaport MS, Stern PH. Parathyroid hormone and calcitonin modify inositol phospholipid metabolism in fetal rat limb bones. J Bone Miner Res 1986;1(2):173–9. [127] Shankar G, Stern PH. Evaluation of the role of second messenger systems in tumor necrosis factor-stimulated resorption of fetal rat limb bones. Bone 1993;14(6):871–6. [128] Stern PH, Stathopoulos VM, Shankar G, Fenton J, 2nd W. Second messengers in thrombin-stimulated bone resorption. J Bone Miner Res 1990;5(5):443–9. [129] Tatrai A, Lee SK, Stern PH. U-73122, a phospholipase C antagonist, inhibits effects of endothelin-1 and parathyroid hormone on signal transduction in UMR-106 osteoblastic cells. Biochim Biophys Acta 1994;1224(3):575–82. [130] Stern PH, Vance DE. Phosphatidylcholine metabolism in neonatal mouse calvaria. Biochem J 1987;244(2):409–15. [131] Stern PH, Shankar G, Fargher RC, Copp DH, Milliken CE, Sato KJ, et al. Salmon stanniocalcin and bovine parathyroid hormone have dissimilar actions on mammalian bone. J Bone Miner Res 1991;6(11):1153–9. [132] Stewart P, Stern P. Calcium/phosphatidylserine-stimulated protein phosphorylation in bone: effect of parathyroid hormone. J Bone Miner Res 1987;2(4):281–7. [133] Stewart PJ, Stathopoulos VM, Stern PH. Parathyroid hormone (1-34) and Nleu8,18Tyr34-parathyroid hormone, (3-34) amide increase diacylglycerol in neonatal mouse calvaria. Horm Metab Res 1991;23(11):535–8. [134] Sanders JL, Stern PH. Expression and phorbol ester-induced down-regulation of protein kinase C isozymes in osteoblasts. J Bone Miner Res 1996;11(12):1862–72. [135] Dossing DA, Radeff JM, Sanders J, Lee SK, Hsieh MR, Stern PH. Parathyroid hormone stimulates translocation of protein kinase C isozymes in UMR-106 osteoblastic osteosarcoma cells. Bone 2001;29(3):223–30. [136] Radeff JM, Nagy Z, Stern PH. Involvement of PKC-beta in PTH, TNF-alpha, and IL-1 beta effects on IL-6 promoter in osteoblastic cells and on PTH-stimulated bone resorption. Exp Cell Res 2001;268(2):179–88. [137] Radeff JM, Singh AT, Stern PH. Role of protein kinase A, phospholipase C and phospholipase D in parathyroid hormone receptor regulation of protein kinase Calpha and interleukin-6 in UMR106 osteoblastic cells. Cell Signal 2004;16(1):105–14. [138] Singh AT, Kunnel JG, Strieleman PJ, Stern PH. Parathyroid hormone (PTH)-(1-34), [Nle(8,18), Tyr34]PTH-(3-34) amide, PTH-(1-31) amide, and PTH-related peptide-(1-34) stimulate phosphatidylcholine hydrolysis in UMR-106 osteoblastic cells: comparison with effects of phorbol 12,13-dibutyrate. Endocrinology 1999;140(1):131–7. [139] Singh AT, Gilchrist A, Voyno-Yasenetskaya T, Radeff-Huang JM, Stern P. H. G alpha12/G alpha13 subunits of heterotrimeric G proteins mediate parathyroid hormone activation of phospholipase D in UMR-106 osteoblastic cells. Endocrinology 2005;146(5):2171–5. [140] Radeff JM, Nagy Z, Stern PH. Rho and Rho kinase are involved in parathyroid hormone-stimulated protein kinase C alpha translocation and IL-6 promoter activity in osteoblastic cells. J Bone Miner Res 2004;19(11):1882–91. [141] Singh AT, Bhattacharyya RS, Radeff JM, Stern PH. Regulation of parathyroid hormone-stimulated phospholipase D in UMR-106 cells by calcium, MAP kinase, and small G proteins. J Bone Miner Res 2003;18(8):1453–60.

I. INTRODUCTION

The Bone World Beyond the Laboratory: ASBMR and NOF

[142] Sanders JL, Stern PH. Protein kinase C involvement in interleukin-6 production by parathyroid hormone and tumor necrosis factor-alpha in UMR-106 osteoblastic cells. J Bone Miner Res 2000;15(5):885–93. [143] Nagy Z, Radeff J, Stern PH. Stimulation of interleukin-6 promoter by parathyroid hormone, tumor necrosis factor alpha, and interleukin-1beta in UMR-106 osteoblastic cells is inhibited by protein kinase C antagonists. J Bone Miner Res 2001;16(7):1220–7. [144] Wang J, Stern PH. Osteoclastogenic activity and RANKL expression are inhibited in osteoblastic cells expressing constitutively active Galpha(12) or constitutively active RhoA. J Cell Biochem 2010;111(6):1531–6. [145] Yoshida T, Clark MF, Stern PH. The small GTPase RhoA is crucial for MC3T3-E1 osteoblastic cell survival. J Cell Biochem 2009;106(5):896–902. [146] Kazmers NH, Ma SA, Yoshida T, Stern PH. Rho GTPase signaling and PTH 3-34, but not PTH 1-34, maintain the actin cytoskeleton and antagonize bisphosphonate effects in mouse osteoblastic MC3T3-E1 cells. Bone 2009;45(1):52–60. [147] Lomri A, Marie PJ. Changes in cytoskeletal proteins in response to parathyroid hormone and 1,25-dihydroxyvitamin D in human osteoblastic cells. Bone Miner 1990;10(1):1–2. [148] Egan JJ, Gronowicz G, Rodan GA. Parathyroid hormone ­promotes the disassembly of cytoskeletal actin and myosin in cultured osteoblastic cells: mediation by cyclic AMP. J Cell ­Biochem 1991;45(1):101–11. [149] Wang J, Gilchrist A, Stern PH. Antagonist minigenes identify genes regulated by parathyroid hormone through G proteinselective and G protein co-regulated mechanisms in osteoblastic cells. Cell Signal 2011;23(2):380–8. [150] Wang J, Stern PH. Dose-dependent differential effects of risedronate on gene expression in osteoblasts. Biochem Pharmacol 2011;81(8):1036–42.

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[151] Wang J, Stern PH. Sex-specific effects of estrogen and androgen on gene expression in human monocyte-derived osteoclasts. J Cell Biochem 2011;112(12):3714–21. [152] Kunnel JG, Igarashi K, Gilbert JL, Stern PH. Bone anabolic responses to mechanical load in vitro involve COX-2 and constitutive NOS. Connect Tissue Res 2004;45(1):40–9. [153] Kunnel JG, Gilbert JL, Stern PH. In vitro mechanical and cellular responses of neonatal mouse bones to loading using a novel micromechanical-testing device. Calcif Tissue Int 2002;71(6):499–507. [154] Yoshida T, Flegler A, Kozlov A, Stern PH. Direct inhibitory and indirect stimulatory effects of RAGE ligand S100 on sRANKLinduced osteoclastogenesis. J Cell Biochem 2009;107(5):917–25. [155] Woo JT, Kasai S, Stern PH, Nagai K. Compactin suppresses bone resorption by inhibiting the fusion of prefusion osteoclasts and disrupting the actin ring in osteoclasts. J Bone Miner Res 2000;15(4):650–62. [156] Woo JT, Kawatani M, Kato M, Shinki T, Yonezawa T, Kanoh N, et al. Reveromycin A, an agent for osteoporosis, inhibits bone resorption by inducing apoptosis specifically in osteoclasts. Proc Natl Acad Sci U S A 2006;103(12):4729–34. [157] Bhattacharyya MH, Whelton BD, Stern PH, Peterson DP. Cadmium accelerates bone loss in ovariectomized mice and fetal rat limb bones in culture. Proc Natl Acad Sci U S A 1988;85(22):8761–5. [158] Witty JP, Foster SA, Stricklin GP, Matrisian LM, Stern PH. Parathyroid hormone-induced resorption in fetal rat limb bones is associated with production of the metalloproteinases collagenase and gelatinase B. J Bone Miner Res 1996;11(1):72–8. [159] Stern PH, Krieger NS, Nissenson RA, Williams RD, Winkler ME, Derynck R, et al. Human transforming growth factor-alpha stimulates bone resorptionin vitro. J Clin Invest 1985;76(5):2016–9. [160] Strewler GJ, Stern PH, Jacobs JW, Eveloff J, Klein RF, Leung SC, et al. Parathyroid hormonelike protein from human renal carcinoma cells. Structural and functional homology with parathyroid hormone. J Clin Invest 1987;80(6):1803–7.

I. INTRODUCTION