1,25(OH)2D3 Induces a Mineralization Defect and Loss of Bone Mineral Density in Genetic Hypercalciuric Stone-Forming Rats

Authors

Adeline H. Ng, Kevin K. Frick, Nancy S. Krieger, John R. Asplin, Madison Cohen-McFarlane, Christopher D. Culbertson, Kelly Kyker-Snowman, Marc D. Grynpas, David A. Bushinsky

Abstract

Genetic hypercalciuric stone-forming (GHS) rats, bred to maximize urine (u) calcium (Ca) excretion, demonstrate increased intestinal Ca absorption, increased bone Ca resorption, and reduced renal Ca reabsorption, all leading to elevated uCa compared to the parental Sprague–Dawley (SD) rats. GHS rats have increased numbers of vitamin D receptors (VDRs) at each site, with normal levels of 1,25(OH)2D3 (1,25D), suggesting their VDR is undersaturated with 1,25D. We have shown that 1,25D induces a greater increase in uCa in GHS than SD rats. To examine the effect of the increased VDR on the osseous response to 1,25D, we fed GHS and SD rats an ample Ca diet and injected either 1,25D [low dose (LD) 12.5 or high dose (HD) 25 ng/100 g body weight/day] or vehicle (veh) daily for 16 days. Femoral areal bone mineral density (aBMD, by DEXA) was decreased in GHS+LD and GHS+HD relative to GHS+veh, while there was no effect on SD. Vertebral aBMD was lower in GHS compared to SD and further decreased in GHS+HD. Both femoral and L6 vertebral volumetric BMD (by μCT) were lower in GHS and further reduced by HD. Histomorphometry indicated a decreased osteoclast number in GHS+HD compared to GHS+veh or SD+HD. In tibiae, GHS+HD trabecular thickness and number increased, with a 12-fold increase in osteoid volume but only a threefold increase in bone volume. Bone formation rate was decreased in GHS+HD relative to GHS+veh, confirming the mineralization defect. The loss of BMD and the mineralization defect in GHS rats contribute to increased hypercalciuria; if these effects persist, they would result in decreased bone strength, making these bones more fracture-prone. The enhanced effect of 1,25D in GHS rats indicates that the increased VDRs are biologically active.

Link To Article

http://dx.doi.org/10.1007/s00223-014-9838-7

Loss of Cbl-PI3K interaction enhances osteoclast survival due to p21-Ras mediated PI3K activation independent of Cbl-b

Authors

Naga Suresh Adapal1, Mary F. Barbe, Alexander Tsygankov, Joseph Lorenzo, Archana Sanjay

Abstract

Cbl family proteins, Cbl and Cbl-b, are E3 ubiquitin ligases and adaptor proteins, which play important roles in bone-resorbing osteoclasts. Loss of Cbl in mice decreases osteoclast migration, resulting in delayed bone development where as absence of Cbl-b decreases bone volume due to hyper-resorptive osteoclasts. A major structural difference between Cbl and Cbl-b is tyrosine 737 (in YEAM motif) only on Cbl, which upon phosphorylation interacts with p85 subunit of phosphatidylinositol-3 Kinase (PI3K). In contrast to Cbl-/- and Cbl-b-/-, mice lacking Cbl-PI3K interaction due to a Y737F (tyrosine to phenylalanine, YF) mutation showed enhanced osteoclast survival, but defective resorption. To investigate whether Cbl-PI3K interaction contributes to distinct roles of Cbl and Cbl-b in osteoclasts, mice bearing CblY737F mutation in the Cbl-b-/- background (YF/YF;Cbl-b-/-) were generated. The differentiation and survival were augmented similarly in YF/YF and YF/YF;Cbl-b-/- osteoclasts, associated with enhanced PI3K signaling suggesting an exclusive role of Cbl-PI3K interaction, independent of Cbl-b. In addition to PI3K, the small GTPase Ras also regulates osteoclast survival. In the absence of Cbl-PI3K interaction, increased Ras GTPase activity and Ras-PI3K binding were observed and inhibition of Ras activation attenuated PI3K mediated osteoclast survival. In contrast to differentiation and survival, increased osteoclast activity observed in Cbl-b-/- mice persisted even after introduction of the resorption-defective YF mutation in YF/YF;Cbl-b-/- mice. Hence, Cbl and Cbl-b play mutually exclusive roles in osteoclasts. Whereas Cbl-PI3K interaction regulates differentiation and survival, bone resorption is predominantly regulated by Cbl-b in osteoclasts.

Link To Article

http://dx.doi.org/10.1002/jcb.24779

Colony-stimulating factor 1 potentiates lung cancer bone metastasis

Authors

Jaclyn Y Hung, Diane Horn, Kathleen Woodruff, Thomas Prihoda, Claude LeSaux, Jay Peters, Fermin Tio and Sherry L Abboud-Werner

Abstract

Colony-stimulating factor 1 (CSF1) is essential for osteoclastogenesis that mediates osteolysis in metastatic tumors. Patients with lung cancer have increased CSF1 in serum and high levels are associated with poor survival. Adenocarcinomas metastasize rapidly and many patients suffer from bone metastasis. Lung cancer stem-like cells sustain tumor growth and potentiate metastasis. The purpose of this study was to determine the role of CSF1 in lung cancer bone metastasis and whether inhibition of CSF1 ameliorates the disease. Human lung adenocarcinoma A549 cells were examined in vitro for CSF1/CSF1R. A549-luc cells were injected intracardiac in NOD/SCID mice and metastasis was assessed. To determine the effect of CSF1 knockdown (KD) in A549 cells on bone metastasis, cells were stably transfected with a retroviral vector containing short-hairpin CSF1 (KD) or empty vector (CT). Results showed that A549 cells express CSF1/CSF1R; CSF1 increased their proliferation and invasion, whereas soluble CSF1R inhibited invasion. Mice injected with A549-luc cells showed osteolytic bone lesions 3.5 weeks after injection and lesions increased over 5 weeks. Tumors recapitulated adenocarcinoma morphology and showed osteoclasts along the tumor/bone interface, trabecular, and cortical bone loss. Analyses of KD cells showed decreased CSF1 protein levels, reduced colony formation in soft agar assay, and decreased fraction of stem-like cells. In CSF1KD mice, the incidence of tumor metastasis was similar to controls, although fewer CSF1KD mice had metastasis in both hind limbs. KD tumors showed reduced CSF1 expression, Ki-67+ cells, and osteoclasts. Importantly, there was a low incidence of large tumors >0.1 mm2 in CSF1KD mice compared with control mice (10% vs 62.5%). This study established a lung osteolytic bone metastasis model that resembles human disease and suggests that CSF1 is a key determinant of cancer stem cell survival and tumor growth. Results may lead to novel strategies to inhibit CSF1 in lung cancer and improve management of bone metastasis.

Link To Article

http://dx.doi.org/10.1038/labinvest.2014.1

Disruption of the anterior–posterior rotator cuff force balance alters joint function and leads to joint damage in a rat model

Authors

Katherine E. Reuther, Stephen J. Thomas, Jennica J. Tucker, Joseph J. Sarver, Chancellor F. Gray, Sarah I. Rooney, David L. Glaser, Louis J. Soslowsky

Abstract

The rotator cuff assists in shoulder movement and provides dynamic stability to the glenohumeral joint. Specifically, the anterior–posterior (AP) force balance, provided by the subscapularis anteriorly and the infraspinatus and teres minor posteriorly, is critical for joint stability and concentric rotation of the humeral head on the glenoid. However, limited understanding exists of the consequences associated with disruption of the AP force balance (due to tears of both the supraspinatus and infraspinatus tendons) on joint function and joint damage. We investigated the effect of disrupting the APforce balance on joint function and joint damage in an overuse rat model. Twenty-eight rats underwent 4 weeks of overuse to produce a tendinopathic condition and were then randomized into two surgical groups: Detachment of the supraspinatus only or detachment of the supraspinatus and infraspinatus tendons. Rats were then gradually returned to their overuse protocol. Quantitative ambulatory measures including medial/lateral, propulsion, braking, and vertical forces were significantly different between groups. Additionally, cartilage and adjacent tendon properties were significantly altered. These results identify joint imbalance as a mechanical mechanism for joint damage and demonstrate the importance of preserving rotator cuff balance when treating active cuff tear patients.

Link To Article

http://dx.doi.org/10.1002/jor.22586

Differential Effects of Collagen Prolyl 3-Hydroxylation on Skeletal Tissues

Authors

Erica P. Homan, Caressa Lietman, Ingo Grafe, Jennifer Lennington, Roy Morello, Dobrawa Napierala, Ming-Ming Jiang, Elda M. Munivez, Brian Dawson, Terry K. Bertin, Yuqing Chen, Rhonald Lua, Olivier Lichtarge, Brendan H. L. Lee

Abstract

Mutations in the genes encoding cartilage associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by PPIB), form a complex that 3-hydroxylates a single proline residue on the α1(I) chain (Pro986) and has cis/trans isomerase (PPIase) activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complex's 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1H662A). This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the α1(I) and α1(II) collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase activity versus complete ablation of the prolyl 3-hydroxylation complex.

Link To Article

http://dx.doi.org/10.1371/journal.pgen.1004121

A closer look at the immediate trabecula response to combined parathyroid hormone and alendronate treatment

Authors

Allison R. Altman, Wei-Ju Tseng, Chantal M.J. de Bakker, Beom Kang Huh, Abhishek Chandra, Ling Qin, X. Sherry Liu

Abstract

Daily injections of parathyroid hormone (PTH) are the only FDA-approved anabolic treatment for osteoporosis; however PTH is only clinically approved for treatment periods of up to 24 months. To enhance its anabolic effect, combining PTH with anti-resorptive therapy was proposed and expected to maximize the effectiveness of PTH. The current study aimed to elucidate structural mechanisms through which combination therapy can further improve bone strength over a limited treatment window of 12 days, to more closely examine the early phase of the anabolic window. We examined 30 female rats treated with either vehicle (Veh), alendronate (ALN), PTH, or both PTH and ALN (PTH + ALN). Standard and individual trabecula segmentation (ITS)-based microstructural analyses were performed using in vivo micro-computed tomography. We found an increase in BV/TV in all treatments with the highest in the PTH + ALN group. Tb.Th* increased in both PTH and PTH + ALN groups well beyond that of the Veh or ALN group. SMI decreased in all treatments with PTH + ALN having the greatest tendency toward plate-like structures. ITS confirmed the trend toward more plate-like structures with increased plate Tb.N and increased plate-to-rod ratio that was most pronounced in the PTH + ALN group. Using image-based finite element analysis, we demonstrated that stiffness increased in all treatment groups, again with the largest increase in the PTH + ALN group, indicating the resulting structural implications of increased plate-like structure. Static and dynamic bone histomorphometry and a serum resorption marker confirmed that PTH + ALN significantly increased bone formation activities and suppressed bone resorption activities. Overall the results indicate that PTH + ALN treatment has an additive effect due to a preferential increase in plate-like structures.

Link To Article

http://dx.doi.org/10.1016/j.bone.2014.01.008