Paul Childress

Anabolic and Catabolic Regimens of Human Parathyroid Hormone 1–34 Elicit Bone- and Envelope-Specific Attenuation of Skeletal Effects in Sost-Deficient Mice

PTH is a potent calcium-regulating factor that has skeletal anabolic effects when administered intermittently or catabolic effects when maintained at consistently high levels. Bone cells express PTH receptors, but the cellular responses to PTH in bone are incompletely understood. Wnt signaling has recently been implicated in the osteo-anabolic response to the hormone. Specifically, the Sost gene, a major antagonist of Wnt signaling, is down-regulated by PTH exposure. We investigated this mechanism by treating Sost-deficient mice and their wild-type littermates with anabolic and catabolic regimens of PTH and measuring the skeletal responses. Male Sost(+/+) and Sost(-/-) mice were injected daily with human PTH 1-34 (0, 30, or 90 μg/kg) for 6 wk. Female Sost(+/+) and Sost(-/-) mice were continuously infused with vehicle or high-dose PTH (40 μg/kg · d) for 3 wk. Dual energy x-ray absorptiometry-derived measures of intermittent PTH (iPTH)-induced bone gain were impaired in Sost(-/-) mice. Further probing revealed normal or enhanced iPTH-induced cortical bone formation rates but concomitant increases in cortical porosity among Sost(-/-) mice. Distal femur trabecular bone was highly responsive to iPTH in Sost(-/-) mice. Continuous PTH (cPTH) infusion resulted in equal bone loss in Sost(+/+) and Sost(-/-) mice as measured by dual energy x-ray absorptiometry. However, distal femur trabecular bone, but not lumbar spine trabecular bone, was spared the bone-wasting effects of cPTH in Sost(-/-) mice. These results suggest that changes in Sost expression are not required for iPTH-induced anabolism. iPTH-induced resorption of cortical bone might be overstimulated in Sost-deficient environments. Furthermore, Sost deletion protects some trabecular compartments, but not cortical compartments, from bone loss induced by high-dose PTH infusion.

Functional Impairment of Bone Formation in the Pathogenesis of Osteoporosis: The Bone Marrow Regenerative Competence

The skeleton is a high-renewal organ that undergoes ongoing cycles of remodeling. The regenerative bone formation arm ultimately declines in the aging, postmenopausal skeleton, but current therapies do not adequately address this deficit. Bone marrow is the primary source of the skeletal anabolic response and the mesenchymal stem cells (MSCs), which give rise to bone matrix-producing osteoblasts. The identity of these stem cells is emerging, but it now appears that the term ‘MSC’ has often been misapplied to the bone marrow stromal cell (BMSC), a progeny of the MSC. Nevertheless, the changes in BMSC phenotype associated with age and estrogen depletion likely contribute to the attenuated regenerative competence of the marrow and may reflect alterations in MSC phenotype. Here we summarize current concepts in bone marrow MSC identity, and within this context, review recent observations on changes in bone marrow population dynamics associated with aging and menopause.

Nmp4/CIZ Suppresses Parathyroid Hormone-Induced Increases in Trabecular Bone

The nucleocytoplasmic shuttling transcription factor Nmp4/CIZ (nuclear matrix protein 4/cas interacting zinc finger protein) is a ubiquitously expressed protein that regulates both cytoplasmic and nuclear activities. In the nucleus, Nmp4/CIZ represses transcription of genes crucial to osteoblast differentiation and genes activated by various anabolic stimuli, including parathyroid hormone (PTH). We investigated the role of Nmp4/CIZ in the PTH‐induced increase in bone by engineering mice with loss‐of‐function mutations in the Nmp4/CIZ gene, and treating 10‐week‐old female mice with anabolic doses of human PTH (1–34) at 30 µg/kg/day, 7 day/week, for 7 weeks or vehicle control. The untreated, baseline phenotype of the Nmp4‐null mice between 8 and 16 weeks of age included a modest but significant increase in bone mineral density (BMD) and bone mineral content (BMC) compared to wild‐type (WT) mice. Type I collagen mRNA expression was moderately elevated in the femurs of the Nmp4‐null mice. The Nmp4 mutant alleles decreased body weight by 4% when expressed on a mixed background but the same alleles on a pure B6 background yielded a significant, 15% increase in body weight among the KO mice, compared to their WT controls. Hormone treatment equally enhanced BMD and BMC over vehicle‐treated mice in both the WT and Nmp4‐null groups but Nmp4‐KO mice exhibited a significantly greater PTH‐induced acquisition of femoral trabecular bone as compared to WT mice. These data support our hypothesis that Nmp4/CIZ is a transcriptional attenuator that suppresses osteoid synthesis and PTH‐mediated acquisition of cancellous bone. J. Cell. Physiol. 219: 734–743, 2009.

Nmp4/CIZ: Road block at the intersection of PTH and load

Teriparatide (parathyroid hormone, [PTH]) is the only FDA-approved drug that replaces bone lost to osteoporosis. Enhancing PTH efficacy will improve cost-effectiveness and ameliorate contraindications. Combining this hormone with load-bearing exercise may enhance therapeutic potential consistent with a growing body of evidence that these agonists are synergistic and share common signaling pathways. Additionally, neutralizing molecules that naturally suppress the anabolic response to PTH may also improve the efficacy of treatment with this hormone. Nmp4/CIZ (nuclear matrix protein 4/cas interacting zinc finger)-null mice have enhanced responses to intermittent PTH with respect to increasing trabecular bone mass and are also immune to disuse-induced bone loss likely by the removal of Nmp4/CIZ suppressive action on osteoblast function. Nmp4/CIZ activity may be sensitive to changes in the mechanical environment of the bone cell brought about by hormone- or mechanical load-induced changes in cell shape and adhesion. Nmp4 was identified in a screen for PTH-responsive nuclear matrix architectural transcription factors (ATFs) that we proposed translate hormone-induced changes in cell shape and adhesion into changes in target gene DNA conformation. CIZ was independently identified as a nucleocytoplasmic shuttling transcription factor associating with the mechano-sensitive focal adhesion proteins p130Cas and zxyin. The p130Cas/zyxin/Nmp4/CIZ pathway resembles the beta-catenin/TCF/LEF1 mechanotransduction response limb and both share features with the HMGB1 (high mobility group box 1)/RAGE (receptor for advanced glycation end products) signaling axis. Here we describe Nmp4/CIZ within the context of the PTH-induced anabolic response and consider the place of this molecule in the hierarchy of the PTH-load response network.

RAGE supports parathyroid hormone-induced gains in femoral trabecular bone

Parathyroid hormone (PTH) restores bone mass to the osteopenic skeleton, but significant questions remain as to the underlying mechanisms. The receptor for advanced glycation end products (RAGE) is a multiligand receptor of the immunoglobulin superfamily; however, recent studies indicate a role in bone physiology. We investigated the significance of RAGE to hormone-induced increases in bone by treating 10-wk-old female Rage-knockout (KO) and wild-type (WT) mice with human PTH-(1-34) at 30 microg.kg(-1).day(-1) or vehicle control, 7 days/wk, for 7 wk. PTH produced equivalent relative gains in bone mineral density (BMD) and bone mineral content (BMC) throughout the skeleton in both genotypes. PTH-mediated relative increases in cortical area of the midshaft femur were not compromised in the null mice. However, the hormone-induced gain in femoral cancellous bone was significantly attenuated in Rage-KO mice. The loss of RAGE impaired PTH-mediated increases in femoral cancellous bone volume, connectivity density, and trabecular number but did not impact increases in trabecular thickness or decreases in trabecular spacing. Disabling RAGE reduced femoral expression of bone formation genes, but their relative PTH-responsiveness was not impaired. Neutralizing RAGE did not attenuate vertebral cancellous bone response to hormone. Rage-null mice exhibited an attenuated accrual rate of bone mass, with the exception of the spine, and an enhanced accrual rate of fat mass. We conclude that RAGE is necessary for key aspects of the skeletons response to anabolic PTH. Specifically, RAGE is required for hormone-mediated improvement of femoral trabecular architecture but not intrinsically necessary for increasing cortical thickness.

Immortalization and characterization of osteoblast cell lines generated from wild-type and Nmp4-null mouse bone marrow stromal cells using murine telomerase reverse transcriptase (mTERT).

Intermittent parathyroid hormone (PTH) adds new bone to the osteoporotic skeleton; the transcription factor Nmp4/CIZ represses PTH‐induced bone formation in mice and as a consequence is a potential drug target for improving hormone clinical efficacy. To explore the impact of Nmp4/CIZ on osteoblast phenotype, we immortalized bone marrow stromal cells from wildtype (WT) and Nmp4‐knockout (KO) mice using murine telomerase reverse transcriptase. Clonal lines were initially chosen based on their positive staining for alkaline phosphatase and capacity for mineralization. Disabling Nmp4/CIZ had no gross impact on osteoblast phenotype development. WT and KO clones exhibited identical sustained growth, reduced population doubling times, extended maintenance of the mature osteoblast phenotype, and competency for differentiating toward the osteoblast and adipocyte lineages. Additional screening of the immortalized cells for PTH‐responsiveness permitted further studies with single WT and KO clones. We recently demonstrated that PTH‐induced c‐fos femoral mRNA expression is enhanced in Nmp4‐KO mice and in the present study we observed that hormone stimulated either an equivalent or modestly enhanced increase in c‐fos mRNA expression in both primary null and KO clone cells depending on PTH concentration. The null primary osteoblasts and KO clone cells exhibited a transiently enhanced response to bone morphogenetic protein 2 (BMP2). The clones exhibited lower and higher expressions of the PTH receptor (Pthr1) and the BMP2 receptor (Bmpr1a, Alk3), respectively, as compared to primary cells. These immortalized cell lines will provide a valuable tool for disentangling the complex functional roles underlying Nmp4/CIZ regulation of bone anabolism. J. Cell. Physiol. 227: 1873–1882, 2012.

Nmp4/CIZ suppresses the parathyroid hormone anabolic window by restricting mesenchymal stem cell and osteoprogenitor frequency

Parathyroid hormone (PTH) anabolic osteoporosis therapy is intrinsically limited by unknown mechanisms. We previously showed that disabling the transcription factor Nmp4/CIZ in mice expanded this anabolic window while modestly elevating bone resorption. This enhanced bone formation requires a lag period to materialize. Wild-type (WT) and Nmp4-knockout (KO) mice exhibited equivalent PTH-induced increases in bone at 2 weeks of treatment, but by 7 weeks, the null mice showed more new bone. At 3-week treatment, serum osteocalcin, a bone formation marker, peaked in WT mice, but continued to increase in null mice. To determine if 3 weeks is the time when the addition of new bone diverges and to investigate its cellular basis, we treated 10-week-old null and WT animals with human PTH (1-34) (30 μg/kg/day) or vehicle before analyzing femoral trabecular architecture and bone marrow (BM) and peripheral blood phenotypic cell profiles. PTH-treated Nmp4-KO mice gained over 2-fold more femoral trabecular bone than WT by 3 weeks. There was no difference between genotypes in BM cellularity or profiles of several blood elements. However, the KO mice exhibited a significant elevation in CFU-F cells, CFU-F(Alk)(Phos+) cells (osteoprogenitors), and a higher percentage of CFU-F(Alk)(Phos+) cells/CFU-F cells consistent with an increase in CD45-/CD146+/CD105+/nestin+ mesenchymal stem cell frequency. Null BM exhibited a 2-fold enhancement in CD8+ T cells known to support osteoprogenitor differentiation and a 1.6-fold increase in CFU-GM colonies (osteoclast progenitors). We propose that Nmp4/CIZ limits the PTH anabolic window by restricting the number of BM stem, progenitor, and blood cells that support anabolic bone remodeling.

Osteomacs interact with megakaryocytes and osteoblasts to regulate murine hematopoietic stem cell function

Networking between hematopoietic stem cells (HSCs) and cells of the hematopoietic niche is critical for stem cell function and maintenance of the stem cell pool. We characterized calvariae-resident osteomacs (OMs) and their interaction with megakaryocytes to sustain HSC function and identified distinguishing properties between OMs and bone marrow (BM)-derived macrophages. OMs, identified as CD45+F4/80+ cells, were easily detectable (3%-5%) in neonatal calvarial cells. Coculture of neonatal calvarial cells with megakaryocytes for 7 days increased OM three- to sixfold, demonstrating that megakaryocytes regulate OM proliferation. OMs were required for the hematopoiesis-enhancing activity of osteoblasts, and this activity was augmented by megakaryocytes. Serial transplantation demonstrated that HSC repopulating potential was best maintained by in vitro cultures containing osteoblasts, OMs, and megakaryocytes. With or without megakaryocytes, BM-derived macrophages were unable to functionally substitute for neonatal calvarial cell-associated OMs. In addition, OMs differentiated into multinucleated, tartrate resistant acid phosphatase-positive osteoclasts capable of bone resorption. Nine-color flow cytometric analysis revealed that although BM-derived macrophages and OMs share many cell surface phenotypic similarities (CD45, F4/80, CD68, CD11b, Mac2, and Gr-1), only a subgroup of OMs coexpressed M-CSFR and CD166, thus providing a unique profile for OMs. CD169 was expressed by both OMs and BM-derived macrophages and therefore was not a distinguishing marker between these 2 cell types. These results demonstrate that OMs support HSC function and illustrate that megakaryocytes significantly augment the synergistic activity of osteoblasts and OMs. Furthermore, this report establishes for the first time that the crosstalk between OMs, osteoblasts, and megakaryocytes is a novel network supporting HSC function.

Uphill running does not exacerbate collagenase-induced pathological changes in the Achilles tendon of rats selectively bred for high-capacity running

Abstract The Achilles tendon is a frequent site for degeneration, and advanced understanding of this pathology requires an animal model that replicates the human condition. The aim of this study was to explore whether intratendinous collagenase injection combined with treadmill running created a pathology in the rat Achilles tendon consistent with human Achilles tendinosis. Collagenase was injected into one Achilles tendon of 88 high-capacity running (HCR) rats, which were randomized into treadmill running and cage control groups. Running animals ran at speeds up to 30 m/min on a treadmill at a 15° incline for up to 1 h/d, 5 d/week for 4 or 10 weeks. Cage control animals maintained cage activity. Collagenase induced molecular, histopathological and mechanical changes within the Achilles tendon at 4 weeks. The mechanical changes persisted at 10 weeks; however, the histopathological and majority of the molecular changes were no longer present at 10 weeks. Treadmill running had minimal effect and did not exacerbate the collagenase-induced changes as there were no statistical interactions between the interventions. These data suggest combined intratendinous collagenase injection and treadmill running does not create pathology within the Achilles tendon of rats selectively bred for HCR that is consistent with human Achilles tendinosis.

Megakaryocyte and Osteoblast Interactions Modulate Bone Mass and Hematopoiesis

Emerging evidence demonstrates that megakaryocytes (MK) play key roles in regulating skeletal homeostasis and hematopoiesis. To test if the loss of MK negatively impacts osteoblastogenesis and hematopoiesis, we generated conditional knockout mice where Mpl, the receptor for the main MK growth factor, thrombopoietin, was deleted specifically in MK (Mplf/f;PF4cre). Unexpectedly, at 12 weeks of age, these mice exhibited a 10-fold increase in platelets, a significant expansion of hematopoietic/mesenchymal precursors, and a remarkable 20-fold increase in femoral midshaft bone volume. We then investigated whether MK support hematopoietic stem cell (HSC) function through the interaction of MK with osteoblasts (OB). LSK cells (Lin-Sca1+CD117+, enriched HSC population) were co-cultured with OB+MK for 1 week (1wk OB+MK+LSK) or OB alone (1wk OB+LSK). A significant increase in colony-forming units was observed with cells from 1wk OB+MK cultures. Competitive repopulation studies demonstrated significantly higher engraftment in mice transplanted with cells from 1wk OB+MK+LSK cultures compared to 1wk OB+LSK or LSK cultured alone for 1 week. Furthermore, single-cell expression analysis of OB cultured±MK revealed adiponectin as the most significantly upregulated MK-induced gene, which is required for optimal long-term hematopoietic reconstitution. Understanding the interactions between MK, OB, and HSC can inform the development of novel treatments to enhance both HSC recovery following myelosuppressive injuries, as well as bone loss diseases, such as osteoporosis.