Abaloparatide prevents immobilization-induced cortical but not trabecular bone loss after spinal cord injury.

Spinal cord injury (SCI) causes severe and resistant sublesional disuse bone loss. Abaloparatide, a modified parathyroid hormone related peptide, is an FDA approved drug for treatment of severe osteoporosis with potent anabolic activity. The effects of abaloparatide on SCI-induced bone loss remain undefined. Thus, female mice underwent sham or severe contusion thoracic SCI causing hindlimb paralysis. Mice then received subcutaneous injection of vehicle or 20 µg/kg/day abaloparatide for 35 days. Micro-computed tomography (micro-CT) analysis of the distal and midshaft femoral regions of the SCI-vehicle mice revealed reduced trabecular fractional bone volume (56%), thickness (75%), and cortical thickness (80%) compared to sham-vehicle controls. Treatment with abaloparatide did not prevent SCI-induced changes in trabecular or cortical bone. However, histomorphometry evaluation of the SCI-abaloparatide mice demonstrated that abaloparatide treatment increased osteoblast (241%) and osteoclast (247%) numbers and the mineral apposition rate (131%) compared to SCI-vehicle animals. In another independent experiment, treatment with 80 µg/kg/day abaloparatide significantly attenuated SCI-induced loss in cortical bone thickness (93%) when compared to SCI-vehicle mice (79%) but did not prevent SCI-induced trabecular bone loss or elevation in cortical porosity. Biochemical analysis of the bone marrow supernatants of the femurs showed that SCI-abaloparatide animals had 2.3-fold increase in procollagen type I N-terminal propeptide, a bone formation marker than SCI-vehicle animals. SCI groups had 70% higher levels of cross-linked C-telopeptide of type I collagen, a bone resorption marker, than sham-vehicle mice. These findings suggest that abaloparatide protects the cortical bone against the deleterious effects of SCI by promoting bone formation.

Inhibition of TGF-ß Signaling Attenuates Disuse-induced Trabecular Bone Loss After Spinal Cord Injury in Male Mice.

Bone loss is one of the most common complications of immobilization after spinal cord injury (SCI). Whether transforming growth factor (TGF)-ß signaling plays a role in SCI-induced disuse bone loss has not been determined. Thus, 16-week-old male mice underwent sham or spinal cord contusion injury to cause complete hindlimb paralysis. Five days later, 10 mg/kg/day control (IgG) or anti-TGF-ß1,2,3 neutralizing antibody (1D11) was administered twice weekly for 4 weeks. Femurs were examined by micro-computed tomography (micro-CT) scanning and histology. Bone marrow (BM) supernatants were analyzed by enzyme-linked immunosorbent assay for levels of procollagen type 1 intact N-terminal propeptide (P1NP), tartrate-resistant acid phosphatase (TRAcP-5b), receptor activator of nuclear factor-kappa B ligand (RANKL), osteoprotegerin (OPG), and prostaglandin E2 (PGE2). Distal femoral micro-CT analysis showed that SCI-1D11 mice had significantly (P < .05) attenuated loss of trabecular fractional bone volume (123% SCI-1D11 vs 69% SCI-IgG), thickness (98% vs 81%), and connectivity (112% vs 69%) and improved the structure model index (2.1 vs 2.7). Histomorphometry analysis revealed that osteoclast numbers were lower in the SCI-IgG mice than in sham-IgG control. Biochemically, SCI-IgG mice had higher levels of P1NP and PGE2 but similar TRAcP-5b and RANKL/OPG ratio to the sham-IgG group. The SCI-1D11 group exhibited higher levels of P1NP but similar TRAcP-5b, RANKL/OPG ratio, and PGE2 to the sham-1D11 group. Furthermore, 1D11 treatment prevented SCI-induced hyperphosphorylation of tau protein in osteocytes, an event that destabilizes the cytoskeleton. Together, inhibition of TGF-ß signaling after SCI protects trabecular bone integrity, likely by balancing bone remodeling, inhibiting PGE2 elevation, and preserving the osteocyte cytoskeleton.

Absence of αß T cells accelerates disuse bone loss in male mice after spinal cord injury.

Whether T cells promote bone loss following immobilization after spinal cord injury (SCI) remains undetermined. Therefore, wild-type (WT) and T cell-deficient (Tcrb-/- ) male mice underwent sham or contusion SCI to cause hindlimb paralysis. Femurs were isolated and distal and midshaft regions were evaluated by microcomputed tomography scanning. Bone marrow (BM) levels of bone turnover markers, as well as receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG), were measured by ELISA. At 2 weeks post-SCI, immobilization resulted in marked reduction in trabecular fractional bone volume (55%), thickness (40%), connectivity, and cortical thickness only in the Tcrb-/- animals (interaction with P < 0.05). BM analysis revealed lower bone formation (procollagen type 1 intact N-terminal propeptide), higher bone resorption (tartrate-resistant acid phosphatase-5b), and a higher RANKL/OPG ratio in the Tcrb-/- SCI animals. At 5 weeks post-SCI, while both WT and Tcrb-/- paralyzed animals showed deterioration of all indices of bone structure, they were more severe in Tcrb-/- animals. In summary, unlike other skeletal disorders, loss of αß T cells compromises, rather than preserves, skeletal integrity under conditions of immobilization.

Effects of a High-Fat Diet on Tissue Mass, Bone, and Glucose Tolerance after Chronic Complete Spinal Cord Transection in Male Mice.

Spinal cord injury (SCI) is associated with obesity and is a risk factor for type 2 diabetes mellitus (T2DM). Immobilization, muscle atrophy, obesity, and loss of sympathetic innervation to the liver are believed to contribute to risks of these abnormalities. Systematic study of the mechanisms underlying SCI-induced metabolic disorders has been limited by a lack of animal models of insulin resistance following SCI. Therefore, the effects of a high-fat diet (HFD), which causes weight gain and glucose intolerance in neurologically intact mice, was tested in mice that had undergone a spinal cord transection at thoracic vertebra 10 (T10) or a sham-transection. At 84 days after surgery, Sham-HFD and SCI-HFD mice showed impaired intraperitoneal glucose tolerance when compared with Sham control (Sham-Con) or SCI control (SCI-Con) mice fed a standard control chow. Glucose tolerance in SCI-Con mice was comparable to that of Sham-Con mice. The mass of paralyzed skeletal muscle, liver, and epididymal, inguinal, and omental fat deposits were lower in SCI versus Sham groups, with lower liver mass present in SCI-HFD versus SCI-Con animals. SCI also produced sublesional bone loss, with no differences between SCI-Con and SCI-HFD groups. The results suggest that administration of a HFD to mice after SCI may provide a model to better understand mechanisms leading to insulin resistance post-SCI, as well as an approach to study pathogenesis of glucose intolerance that is independent of obesity.

Abaloparatide exhibits greater osteoanabolic response and higher cAMP stimulation and ß-arrestin recruitment than teriparatide.

Teriparatide and abaloparatide are parathyroid hormone receptor 1 (PTHR1) analogs with unexplained differential efficacy for the treatment of osteoporosis. Therefore, we compared the effects of abaloparatide and teriparatide on bone structure, turnover, and levels of receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG). Wild-type (WT) female mice were injected daily with vehicle or 20-80 µg/kg/day of teriparatide or abaloparatide for 30 days. Femurs and spines were examined by microcomputed tomography scanning and serum levels of bone turnover markers, RANKL, and OPG, were measured by ELISA. Both analogs similarly increased the distal femoral fractional trabecular bone volume, connectivity, and number, and reduced the structure model index (SMI) at 20-80 µg/kg/day doses. However, only abaloparatide exhibited a significant increase (13%) in trabecular thickness at 20 µg/kg/day dose. Femoral cortical evaluation showed that abaloparatide caused a greater dose-dependent increase in cortical thickness than teriparatide. Both teriparatide and abaloparatide increased lumbar 5 vertebral trabecular connectivity but had no or modest effect on other indices. Biochemical analysis demonstrated that abaloparatide promoted greater elevation of procollagen type 1 intact N-terminal propeptide, a bone formation marker, and tartrate-resistant acid phosphatase 5b levels, a bone resorption marker, and lowered the RANKL/OPG ratio. Furthermore, PTHR1 signaling was compared in cells treated with 0-100 nmol/L analog. Interestingly, abaloparatide had a markedly lower EC50 for cAMP formation (2.3-fold) and ß-arrestin recruitment (1.6-fold) than teriparatide. Therefore, abaloparatide-improved efficacy can be attributed to enhanced bone formation and cortical structure, reduced RANKL/OPG ratio, and amplified Gs-cAMP and ß-arrestin signaling.

Key Glycolytic Metabolites in Paralyzed Skeletal Muscle Are Altered Seven Days after Spinal Cord Injury in Mice.

Spinal cord injury (SCI) results in rapid muscle atrophy and an oxidative-to-glycolytic fiber-type shift. Those with chronic SCI are more at risk for developing insulin resistance and reductions in glucose clearance than able-bodied individuals, but how glucose metabolism is affected after SCI is not well known. An untargeted metabolomics approach was utilized to investigate changes in whole-muscle metabolites at an acute (7-day) and subacute (28-day) time frame after a complete T9 spinal cord transection in 20-week-old female C57BL/6 mice. Two hundred one metabolites were detected in all samples, and 83 had BinBase IDs. A principal components analysis showed the 7-day group as a unique cluster. Further, 36 metabolites were altered after 7- and/or 28-day post-SCI (p values <0.05), with 12 passing further false discovery rate exclusion criteria; of those 12 metabolites, three important glycolytic molecules-glucose and downstream metabolites pyruvic acid and lactic acid-were reduced at 7 days compared to those values in sham and/or 28-day animals. These changes were associated with altered expression of proteins associated with glycolysis, as well as monocarboxylate transporter 4 gene expression. Taken together, our data suggest an acute disruption of skeletal muscle glucose uptake at 7 days post-SCI, which leads to reduced pyruvate and lactate levels. These levels recover by 28 days post-SCI, but a reduction in pyruvate dehydrogenase protein expression at 28 days post-SCI implies disruption in downstream oxidation of glucose.

Daily parathyroid hormone administration enhances bone turnover and preserves bone structure after severe immobilization-induced bone loss.

Immobilization, as a result of motor-complete spinal cord injury (SCI), is associated with severe osteoporosis. Whether parathyroid hormone (PTH) administration would reduce bone loss after SCI remains unclear. Thus, female mice underwent sham or surgery to produce complete spinal cord transection. PTH (80 µg/kg) or vehicle was injected subcutaneously (SC) daily starting on the day of surgery and continued for 35 days. Isolated tibias and femurs were examined by microcomputed tomography scanning (micro-CT) and histology and serum markers of bone turnover were measured. Micro-CT analysis of tibial metaphysis revealed that the SCI-vehicle animals exhibited 49% reduction in fractional trabecular bone volume and 18% in trabecular thickness compared to sham-vehicle controls. SCI-vehicle animals also had 15% lower femoral cortical thickness and 16% higher cortical porosity than sham-vehicle counterparts. Interestingly, PTH administration to SCI animals restored 78% of bone volume, increased connectivity to 366%, and lowered structure model index by 10% compared to sham-vehicle animals. PTH further favorably attenuated femoral cortical bone loss to 5% and prevented the SCI-associated cortical porosity. Histomorphometry evaluation of femurs of SCI-vehicle animals demonstrated a marked 49% and 38% decline in osteoblast and osteoclast number, respectively, and 35% reduction in bone formation rate. In contrast, SCI-PTH animals showed preserved osteoblast and osteoclast numbers and enhanced bone formation rate. Furthermore, SCI-PTH animals had higher levels of bone formation and resorption markers than either SCI- or sham-vehicle groups. Collectively, these findings suggest that intermittent PTH receptor activation is an effective therapeutic strategy to preserve bone integrity after severe immobilization.

Activation of Protein Kinase A in Mature Osteoblasts Promotes a Major Bone Anabolic Response.

Protein kinase A (PKA) regulates osteoblast cell function in vitro and is activated by important bone mass modulating agents. We determined whether PKA activation in osteoblasts is sufficient to mediate a bone anabolic response. Thus, a mouse model conditionally expressing a constitutively active PKA (CA-PKA) in osteoblasts (CA-PKA-OB mouse) was developed by crossing a 2.3-kb α1 (I)-collagen promoter-Cre mouse with a floxed-CA-PKA mouse. Primary osteoblasts from the CA-PKA-OB mice exhibited higher basal PKA activity than those from control mice. Microcomputed tomographic analysis revealed that CA-PKA-OB female mice had an 8.6-fold increase in femoral but only 1.16-fold increase in lumbar 5 vertebral bone volume/total volume. Femur cortical thickness and volume were also higher in the CA-PKA-OB mice. In contrast, alterations in many femoral microcomputed tomographic parameters in male CA-PKA-OB mice were modest. Interestingly, the 3-dimensional structure model index was substantially lower both in femur and lumbar 5 of male and female CA-PKA-OB mice, reflecting an increase in the plate to rod-like structure ratio. In agreement, femurs from female CA-PKA-OB mice had greater load to failure and were stiffer compared with those of control mice. Furthermore, the CA-PKA-OB mice had higher levels of serum bone turnover markers and increased osteoblast and osteoclast numbers per total tissue area compared with control animals. In summary, constitutive activation of PKA in osteoblasts is sufficient to increase bone mass and favorably modify bone architecture and improve mechanical properties. PKA activation in mature osteoblasts is, therefore, an important target for designing anabolic drugs for treating diseases with bone loss.

Disruption of parathyroid hormone and parathyroid hormone-related peptide receptor phosphorylation prolongs ERK1/2 MAPK activation and enhances c-fos expression.

Previous studies have demonstrated that parathyroid hormone (PTH) binding to the PTH/PTH-related peptide receptor (PPR) stimulates G protein coupling, receptor phosphorylation, ß-arrestin translocation, and internalization of the ligand/receptor complex. The extracellular signal-regulated mitogen-activated protein kinases 1/2 (ERK1/2 MAPK) are downstream effectors of PPR. In the current study, we investigated the role of PPR phosphorylation in the PTH regulation of the ERK1/2 MAPK pathway. Short treatment with PTH (0-40 min) of LLCP-K(1) cells stably expressing a wild-type (WT) or a phosphorylation-deficient (PD) PPR (WT-PPR or PD-PPR cells, respectively) results in similar activation of ERK1/2. Interestingly, PTH stimulation of ERK1/2 in the WT-PPR cells then decreases as a result of longer PTH (60 min) treatment, and inhibition of ERK1/2 by PTH is observed at 90 min. Strikingly, the PD-PPR cells exhibit prolonged ERK1/2 activation up to 90 min of PTH treatment. An ERK1/2-dependent increase in c-fos expression is observed in the PD-PPR cells. Subsequently, c-fos expression in the WT-PPR and PD-PPR cells was markedly attenuated by a specific ERK1/2 pathway inhibitor. Further investigations revealed that PTH treatment causes a robust recruitment of a green fluorescent protein-tagged ß-arrestin2 (ß-arrestin2-GFP) in the WT-PPR cells. In contrast, ß-arrestin2 recruitment was reduced in the PD-PPR cells. Importantly, expression of a receptor phosphorylation-independent ß-arrestin2 (R169E) in the PD-PPR cells restored the biphasic effect of PTH on ERK1/2 as in the WT-PPR cells. The study reports a novel role for receptor phosphorylation and ß-arrestin2 in the subsequent inhibition of the ERK1/2 pathway and in control of gene expression.

Extralarge XL(alpha)s (XXL(alpha)s), a variant of stimulatory G protein alpha-subunit (Gs(alpha)), is a distinct, membrane-anchored GNAS product that can mimic Gs(alpha).

GNAS gives rise to multiple imprinted gene products, including the alpha-subunit of the stimulatory G protein (Gs(alpha)) and its variant XL(alpha)s. Based on genomic sequence, the translation of XL(alpha)s begins from the middle of a long open reading frame, suggesting the existence of an N-terminally extended variant termed extralarge XLalphas (XXL(alpha)s). Although XXL(alpha), like Gs(alpha) and XL(alpha)s, would be affected by most disease-causing GNAS mutations, its authenticity and biological significance remained unknown. Here we identified a mouse cDNA clone that comprises the entire open reading frame encoding XXL(alpha)s. Whereas XXL(alpha)s mRNA was readily detected in mouse heart by RT-PCR, it appeared virtually absent in insulinoma-derived INS-1 cells. By Northern blots and RT-PCR, XXL(alpha)s mRNA was detected primarily in the mouse brain, cerebellum, and spleen. Immunohistochemistry using a specific anti-XXL(alpha)s antibody demonstrated XXL(alpha)s protein in multiple brain areas, including dorsal hippocampus and cortex. In transfected cells, full-length human XXL(alpha)s was localized to the plasma membrane and mediated isoproterenol- and cholera toxin-stimulated cAMP accumulation. XXL(alpha)s-R844H, which bears a mutation analogous to that in the constitutively active Gs(alpha) mutant Gs(alpha)-R201H (gsp oncogene), displayed elevated basal signaling. However, unlike Gs(alpha)-R201H, which mostly remains in the cytoplasm, both XXL(alpha)s-R844H and a constitutively active XL(alpha)s mutant localized to the plasma membrane. Hence, XXL(alpha)s is a distinct GNAS product and can mimic Gs(alpha), but the constitutively active XXL(alpha)s and Gs(alpha) mutants differ from each other regarding subcellular targeting. Our findings suggest that XXL(alpha)s deficiency or hyperactivity may contribute to the pathogenesis of diseases caused by GNAS mutations.

Negative regulation of parathyroid hormone (PTH)-activated phospholipase C by PTH/PTH-related peptide receptor phosphorylation and protein kinase A.

PTH binding to the PTH/PTHrP receptor activates adenylate cyclase/protein kinase A (PKA) and phospholipase C (PLC) pathways and increases receptor phosphorylation. The mechanisms regulating PTH activation of PLC signaling are poorly understood. In the current study, we explored the role of PTH/PTHrP receptor phosphorylation and PKA in PTH activation of PLC. When treated with PTH, LLCPK-1 cells stably expressing a green fluorescent protein (GFP)-tagged wild-type (WT) PTH/PTHrP receptor show a small dose-dependent increase in PLC signaling as measured by inositol trisphosphate accumulation assay. In contrast, PTH treatment of LLCPK-1 cells stably expressing a GFP-tagged receptor mutated in its carboxyl-terminal tail so that it cannot be phosphorylated (PD-GFP) results in significantly higher PLC activation (P<0.001). The effects of PTH on PLC activation are dose dependent and reach maximum at the 100 nm PTH dose. When WT receptor-expressing cells are pretreated with H89, a specific inhibitor of PKA, PTH activation of PLC signaling is enhanced in a dose-dependent manner. H89 pretreatment in PD-GFP cells causes a further increase in PLC activation in response to PTH treatment. Interestingly, PTH and forskolin (adenylate cyclase/PKA pathway activator) treatment causes an increase in PLCbeta3 phosphorylation at the Ser1105 inhibitory site and that increase is blocked by the PKA inhibitor, H89. Expression of a mutant PLCbeta3 in which Ser1105 was mutated to alanine (PLCbeta3-SA), in WT or PD cells increases PTH stimulation of inositol 1,4,5-trisphosphate formation. Altogether, these data suggest that PTH signaling to PLC is negatively regulated by PTH/PTHrP receptor phosphorylation and PKA. Furthermore, phosphorylation at Ser1105 is demonstrated as a regulatory mechanism of PLCbeta3 by PKA.

Important role for the V-type H(+)-ATPase and the Golgi apparatus in the recycling of PTH/PTHrP receptor.

Our previous studies demonstrated that a green fluorescent protein-tagged parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor stably expressed in LLCPK-1 cells undergoes agonist-dependent internalization into clathrin-coated pits. The subcellular localization of the internalized PTH/PTHrP receptor is not known. In the present study, we explored the intracellular pathways of the internalized PTH/PTHrP receptor. Using immunofluorescence and confocal microscopy, we show that the internalized receptors localize at a juxtanuclear compartment identified as the Golgi apparatus. The receptors do not colocalize with lysosomes. Furthermore, whereas the internalized receptors exhibit rapid recycling, treatment with proton pump inhibitors (bafilomycin-A1 and concanamycin A) or brefeldin A, Golgi disrupting agents, reduces PTH/PTHrP receptor recycling. Together, these data indicate an important role for the vacuolar-type hydrogen-ATPase and the Golgi apparatus in postendocytic PTH/PTHrP receptor recovery.

Phosphorylation of the receptor for PTH and PTHrP is required for internalization and regulates receptor signaling.

We have previously shown that agonist-dependent phosphorylation of the PTH/PTHrP receptor occurs on its carboxyl-terminal tail. Using site-directed mutagenesis, phosphopeptide mapping, and direct sequencing of cyanogen bromide-cleaved fragments of phosphoreceptors, we report here that PTH-dependent phosphorylation occurs on the serine residues at positions 491, 492, 493, 495, 501, and 504, and that the serine residue at position 489 is required for phosphorylation. When these seven sites were mutated to alanine residues, the mutant receptor was no longer phosphorylated after PTH stimulation. The phosphorylation-deficient receptor, stably expressed in LLCPK-1 cells, was impaired in PTH-dependent internalization and showed an increased sensitivity to PTH stimulation; the EC(50) for PTH-stimulated cAMP accumulation was decreased by 7-fold. Furthermore, PTH stimulation of the phosphorylation-deficient PTH/PTHrP receptor caused a sustained elevation in intracellular cAMP levels. These data indicate that agonist-dependent phosphorylation of the PTH/PTHrP receptor plays an important role in receptor function.