Introduction
Parathyroid hormone (PTH) and PTH-related protein (PTHrP) have a common G protein-coupled receptor, PTH1R, which plays a pivotal role in bone turnover and calcium homeostasis [
1]. Numerous studies have revealed that PTH and PTHrP increased bone mass due to the greater acceleration of bone formation than resorption when administrated intermittently [
2,
3]. In contrast, continuous infusion caused bone loss and hypercalcemia because bone resorption was predominantly promoted over formation [
4,
5]. These findings suggest that PTH and PTHrP are useful for the treatment of certain kinds of bone disease such as osteoporosis when they are administrated appropriately.
In fact, teriparatide (TPTD), the N-terminal 34 amino acids fragment of PTH, is currently the only bone anabolic agent used for the treatment of osteoporosis, by daily or weekly subcutaneous administration [
6,
7]. The effect of TPTD on bone mineral density (BMD) is greater than antiresorptive agents such as bisphosphonates. However, its effect on the hip is modest and the usage is limited in patients with the risk of hypercalcemia because blood calcium is mildly increased by the stimulation of bone resorption.
The mechanism of action of TPTD on bone turnover is not fully understood; however, many studies have examined the molecular mechanisms in recent decades. According to a previous study, PTH(1–34) acts on osteoblasts and indirectly induces bone resorption by activating osteoclasts [
8]. PTH(1–34) increases the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) and decreases the expression of osteoprotegerin (OPG), a decoy receptor of RANKL [
9]. These effects of PTH(1–34) further promote the maturation of osteoclasts with bone resorbing activity. On the other hand, PTH(1–34) promotes the differentiation and mineralization of osteoblasts. This anabolic action of PTH(1–34) is thought to be mediated in part by the enhancement of insulin-like growth factor-1 (IGF-1) expression [
10]. Moreover, it has recently been reported that PTH(1–34) also affects osteocytes, the terminal differentiation state of osteoblasts, and that it exerts anabolic action by inhibiting the secretion of WNT signaling antagonists such as sclerostin and dickkopf-related protein 1 (DKK1) [
11,
12]. These effects of PTH(1–34) are induced mainly by intracellular cAMP, which is generated after the ligand binds to PTH1R [
13].
Abaloparatide (ABL), a novel synthetic peptide analog of PTHrP, has recently approved by Food and Drug Administration as a drug for severe osteoporosis. In the phase 3 ACTIVE clinical trial, daily subcutaneous administration of ABL showed robust BMD increases at the hip as well as the spine, and resulted in a reduced incidence of vertebral and non-vertebral fractures, whereas its calcemic effect was reduced as compared with TPTD [
14]. Analysis of bone turnover markers also suggested that ABL increased both bone formation and resorption, although its effects on both makers of bone formation and resorption were attenuated as compared with TPTD [
14]. These results suggest that ABL is an anabolic agent in which bone formation is predominantly enhanced. However, why the effect on bone turnover is different between these peptides remains unclear. Although there were some reports on bone anabolic effects of ABL in non-clinical studies [
15‐
17], none of them have evaluated in head-to-head comparison with TPTD.
In the present study, we evaluated the bone anabolic effects of ABL and TPTD by intermittent administration to ovariectomized (OVX) rats. Furthermore, to elucidate the difference between ABL and TPTD on bone turnover, the expression of bone formation and resorption-related factors were evaluated in osteoblastic cells by transient or intermittent treatment.
Materials and Methods
Peptides and Cells
Abaloparatide ([Glu22,25, Leu23,28,31, Aib29, Lys26,30] human PTHrP(1–34)–NH2) was synthesized by IPSEN (Paris, France). Teriparatide (human PTH(1–34)) was purchased from BACHEM (Bubendorf, Switzerland).
Cell Culture
The human osteoblastic cell line SaOS-2 and the rat osteoblastic cell line UMR-106 were purchased from the European Collection of Cell Cultures (Wiltshire, UK). SaOS-2 and UMR-106 were maintained in growth medium (GM): McCoy’s 5A (for SaOS-2) or Dulbecco’s Modified Eagle’s Medium (for UMR-106) supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO2. To initiate cell differentiation, cells were grown in differentiation medium (DM): GM containing 50 µg/mL ascorbic acid, 10 mmol/L β-glycerophosphate, and 10 nmol/L dexamethasone. The culture medium was exchanged every 6 days or less.
cAMP Accumulation Assay
Cells were cultured in 96-well plates at a density of 4 × 104 cells/well in GM overnight. The medium was replaced with serum-free GM (containing 0.1% BSA instead of 10% FBS) for culture under starvation conditions. Twenty-four hours after the start of starvation, ABL, TPTD, or vehicle (water containing 0.1% BSA) were added to the cells with 2 mmol/L of 3-isobutyl-1-methylxanthine for 10 min. Cells were lysed and accumulated cAMP was measured using the cAMP-Screen System (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions.
Animal Experiment
Ten-week-old female Sprague–Dawley rats were purchased from Charles River Laboratories Japan, Inc. (Kanagawa, Japan) and ovariectomized at 12 weeks old. Thirty-five days after surgery, rats received once daily subcutaneous injection of ABL, TPTD, or vehicle (saline containing 0.1% heat-inactivated rat serum) for 28 days. Day 0 indicates the day when administration was started. On day 26, rats were anesthetized with intraperitoneally administered pentobarbital sodium and the lumbar spine (L4 and L5) BMD was measured with a PIXImus2 (GE Healthcare, Chicago, IL). Blood (collected from the tail vein) and urine samples were obtained on day 28. Prior to collection, rats were fasted for 15–17 h. Rats were euthanized after the blood and urine collection, and the lumbar spine was removed for the measurement of bone strength. All experimental procedures were approved by the Animal Care and Use Committee of Teijin Institute for Bio-Medical Research.
Measurement of Bone Turnover Markers
Serum P1NP concentration was measured with a Rat/Mouse PINP EIA kit (Immunodiagnostic Systems, Boldon, UK). Urine DPD concentration was measured with an Osteolinks DPD kit (Quidel, San Diego, CA, USA). Urine creatinine (Cr) concentration was measured with a 7180 Autoanalyzer (Hitachi High-Technologies Corporation, Tokyo, Japan).
Measurement of Bone Strength
L4 was isolated from the lumbar spine. The height of the vertebral body was measured with a vernier caliper. To obtain vertebral body specimens, the cranial and caudal ends of the vertebral body were cut with a diamond saw to a height of 4 mm. Bone strength was measured with a MZ-500S (MARUTO Testing Machine Company, Tokyo, Japan).
Evaluation of Bone Resorption-Related Factors
SaOS-2 was cultured in 48-well plates at a density of 5 × 104 cells/well in DM for 10 days. The medium was replaced with serum-free DM for culture under starvation conditions. Twenty-four hours after the start of starvation, cells were treated with ABL and TPTD at a concentration of 100 nmol/L. Six hours after treatment, cells were washed with PBS twice, and cultured with peptide-free or peptide-containing medium for the rest of the experimental period.
SaOS-2 was cultured in 12-well plates at a density of 2 × 105 cells/well in DM. Concomitant with the initiation of cell differentiation, cells were treated with 100 nmol/L of ABL and TPTD once daily for 11 days. The peptide exposure was limited to the first 6 h of the 24-h incubation cycle, and the medium was replaced with peptide-free medium for the rest of the cycle after washing with PBS twice.
Evaluation of WNT Signaling Antagonists
SaOS-2 was cultured in 48-well plates at a density of 5 × 104 cells/well in DM for 30 days. Cells were treated with ABL and TPTD at a concentration of 100 nmol/L. Six hours after treatment, cells were washed with PBS twice, and cultured with peptide-free or peptide-containing medium for the rest of the experimental period.
Quantitative RT-PCR
Cells were lysed and total RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). RNA was reverse-transcribed into cDNA using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA). Quantitative RT-PCR was performed on a 7500 Real-Time PCR System (Applied Biosystems) using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The primers used in this study were as follows: 5′-CGATGGTGGATGGCTCATG-3′ (forward) and 5′-ACCAGATGGGATGTCGGTG-3′ (reverse) for RANKL, 5′-GATGTGGTGACCAAGCCTGA-3′ (forward) and 5′-CTCAGAGTCCTCCCAGGTCA-3′ (reverse) for M-CSF, 5′-GCCTGGCACCAAAGTAAACG-3′ (forward) and 5′-GCTCGAAGGTGAGGTTAGCA-3′ (reverse) for OPG, 5′-TTTCAAGCCACCCATTGACC-3′ (forward) and 5′-GCGGGTACAAGATAAATATCCAAAC-3′ (reverse) for IGF1, 5′-CACCGAGACACCATGAGAGC-3′ (forward) and 5′-CTGCTTGGACACAAAGGCTGC-3′ (reverse) for osteocalcin, 5′-CCTGTGCTCTCCCAGTAACC-3′ (forward) and 5′-CTTCATTTGCCAAGGGTGGTG-3′ (reverse) for DMP1, 5′-TGGCAGGCGTTCAAGAATGA-3′ (forward) and 5′-TGTACTCGGACACGTCTTTGG-3′ (reverse) for SOST, 5′-TGACAACTACCAGCCGTACC-3′ (forward) and 5′-CAGGCGAGACAGATTTGCAC-3′ (reverse) for DKK1, 5′-GTGAAGGTCGGAGTCAACG-3′ (forward) and 5′-TGAGGTCAATGAAGGGGTC-3′ (reverse) for GAPDH.
Measurement of M-CSF, Sclerostin and DKK1
Human Quantikine M-CSF, Sclerostin, and DKK1 ELISA kits (R&D Systems, Minneapolis, MN, USA) were used to quantify M-CSF, sclerostin, and DKK1, respectively, in the cell supernatants according to the manufacturer’s instructions.
Data Analysis
All data are expressed as means ± s.e.m. For calculation of EC50 and Emax, 4-parameter logistic curve fitting was performed. Student’s t test was used for two-group comparisons. Dunnett’s or Tukey’s test was used for multiple comparisons. Significance was inferred from P values of < 0.05. All data were analyzed using GraphPad Prism version 6.03 (GraphPad Software, La Jolla, CA, USA).
Discussion
In the present study, we evaluated the effects of ABL on bone anabolism and bone turnover as compared with TPTD. ABL increased cAMP accumulation in osteoblastic cells, and also increased BMD in OVX rats by intermittent administration similar to TPTD. The effects of ABL on bone were suggested to be anabolic, as evidenced by the increased serum P1NP. These results were consistent with the clinical finding that this peptide had potent efficacy in patients with osteoporosis.
Unlike the clinical study [
14], the maker of bone resorption was poorly affected by both peptides in our OVX experiment, which made us difficult to compare the influence on bone turnover. One major possible reason for the difference is due to the use of young rats in our study. In contrast to postmenopausal women with osteoporosis, young rats continue to growth and show the high level of bone remodeling. These profiles of young rats may mask the effect of the two peptides on bone resorption. Another reason may be the short duration of the treatment period or insufficient treatment frequency optimized for rodents. Bone histomorphometrical analysis may be required to detect and compare the bone resorption effect between ABL and TPTD in this study.
We therefore used human osteoblastic cells to evaluate the effects on bone resorption- and formation-related factors in vitro. We found that the effects of ABL on bone resorption-related factors were significantly attenuated when the treatment was limited to the first 6 h as compared with TPTD. These results suggest that ABL stimulates bone resorption less than TPTD under transient treatment; however, the influence of the two peptides on osteoclastogenesis requires elucidation.
It is unclear why ABL stimulates lower expression of bone resorption-related factors than TPTD with transient treatment only. Recently, it was reported that PTH1R has at least two types of active conformations and that the binding conformation selectivity differs between ABL and TPTD [
21,
22]. This difference further affects the duration of the downstream signaling. As a result, cAMP production by TPTD is sustained after the unbound ligand is washed out, while that by ABL is transient [
22]. We therefore thought that the difference in bone resorption-related factors could arise from the duration of cAMP elevation between the two peptides.
There is another possibility that the two peptides could show the different activity on protein kinase C (PKC). PKC is the downstream signal of PTH1R and is involved in the stimulation of bone resorption-related factors and bone catabolism [
23]. In fact, ostabolin, an amidated form of human PTH(1–31), differs from PTH(1–34) in PKC activation [
24]. It showed cAMP accumulation without activating PKC, while PTH(1–34) stimulated both cAMP and PKC [
24]. The effects of ostabolin on bone turnover look like that of ABL to some extent; ostabolin stimulated bone formation similar to PTH(1–34), with less effect on bone resorption than PTH(1–34) in the preclinical study [
25]. Further research would be required on the effect of ABL on PKC activation for fully understanding of mechanism of action.
It should be noted that there were no significant differences in IGF-1 and osteocalcin expression between the two peptides following the 6-h intermittent treatment. Similarly, the effects of the two peptides on sclerostin and DKK1 expression were almost the same regardless of the exposure time. These results suggest that the effects on bone formation-related factors and WNT signaling inhibitors are comparable between ABL and TPTD even with intermittent/transient treatment, unlike those on bone resorption-related factors.
A different regulation system appears to exist between the bone resorption- and formation-related factors. One hypothesis is that these factors are regulated by the balance between the duration and intensity of cAMP signaling. In other words, the bone resorption-related factors were solely dependent on the duration of cAMP generation, while the bone formation-related factors including WNT signaling inhibitors were affected by the amount of cAMP rather than duration. Notably, mice injected with a PTH analog modified to prolong cAMP generation exhibited highly increased bone resorption and blood calcium concentration [
26]. Further research will be required to clarify whether downstream signaling was influenced by the shifting balance between cAMP duration and intensity.
The half-life of ABL and TPTD in humans was approximately an hour or less following subcutaneous injection [
27,
28], suggesting almost all of the peptides were degraded in the first several hours. It is likely that our results using transient/intermittent 6-h treatment reflect the outcome of the clinical study. In the ph3 ACTIVE study, subcutaneous injection of ABL increased the bone formation marker with less stimulation of the bone resorption marker than observed for TPTD [
14].
In conclusion, our study demonstrated that ABL increased BMD as well as bone strength by enhancing bone formation similar to TPTD. It was also suggested that ABL stimulated the expression of RANKL/OPG and M-CSF less than TPTD under transient 6-h treatment, while bone formation-related factors and WNT signaling inhibitors were similarly enhanced. The profile of ABL indicates that it would be a suitable bone anabolic agent for osteoporosis.
Compliance with Ethical Standards