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Bone 122 (2019) 123–135 Contents lists available at ScienceDirect Bone journal homepage: www.elsevier.com/locate/bone Full Length Article Development and characterization of supramolecular calcitonin assembly and assessment of its interactions with the bone remodelling process T Kapil Manglania, Viji Vijayana, Chandramani Pathakb, Mayuri Khandelwala, Parminder Singhc, ⁎ Stalin Chellappaa, Vijay K. Yadavc, Avadhesha Suroliad, Sarika Guptaa, a Molecular Science Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India b Cell Biology Laboratory, School of Biological Sciences and Biotechnology, Indian Institute of Advanced Research, Gandhinagar 382007, Gujarat, India c Metabolic Research Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India d Molecular Biophysics Unit, Indian Institute of Sciences, Bengaluru 560012, Karnataka, India ARTICLE INFO ABSTRACT Keywords: Osteoporosis is the most common metabolic bone disease, which poses an immense socio-economic burden on Human calcitonin the society. Human calcitonin, though safe, is not considered as a therapeutic option because of its high tendency Protein aggregation to self-associate to form amyloid fibrils thereby affecting its potency. To circumvent this issue we harnessed the Ovariectomy inherent capacity of aggregation and developed an assemblage of human calcitonin monomers, [Supramolecular Osteoporosis Calcitonin Assembly (SCAeI)], which releases biologically active calcitonin monomers in a sustained manner for a period of at least three weeks. AFM and FT-IR analysis showed that SCA-I is amorphous aggregates of calcitonin monomers. Both SCA-I and monomer released from it demonstrated superior anti-osteoclast activity and pro- teolytic stability in-vitro. SCA-I upon single injection significantly improved bone formation markers and reduced bone resorption markers in ovariectomized (OVX) rat model of postmenopausal osteoporosis. Micro-CT analysis revealed that calcitonin released from SCA-I exhibits its beneficial effect on cortical bone more profoundly compared to trabecular bone. This study demonstrates that SCA-I is more effective compared to the human calcitonin monomers on osteoclasts and has site-specificeffect on bone in a model of post-menopausal osteo- porosis. This approach opens up an innovative way to use and study the function of human calcitonin. 1. Introduction 50 times more potent than human calcitonin and possesses superior analgesic effects [7,8]. Several modes of administration have been de- Osteoporosis, a most common disorder of bone remodelling, is veloped such as injectable, nasal spray and oral formulations. Nasal and growing as a major public health problem in the elderly as well as oral routes are not preferred due to poor absorption, high variability young population [1]. Osteoporosis is common in both men and women and rapid degradation by proteases and enzymes [9,10]. Subcutaneous but post-menopausal women are more susceptible to bone loss [2]. Till and intramuscular injections are associated with the drawbacks of poor date several therapeutic interventions have been approved for treat- patient compliance due to the need of multiple injections. Though re- ment of the disease. Most of them are anti-resorptive agents that target search on the anti-resorptive potential of human calcitonin has been osteoclast activity which include bisphosphonates, estrogen replace- initiated in 1960s but still remained challenging due to the inherent ment therapies, selective estrogen receptor modulators (SERMs), cal- tendency of the hormone to associate, fibrillize and lose potency [11]. citonin and strontium ranelate [3]. Calcitonin has been approved as an Bioavailability and stability of human calcitonin prevents its way of alternative to hormone replacement therapy or alendronate for patients being the first line therapy to osteoporosis. Therefore, a novel mode of who have found other treatments unsuccessful or difficult to tolerate drug delivery is prerequisite which could eliminate the shortcomings [4,5]. associated with bioavailability, potency and multiple injections. Calcitonin is a 32 amino acid peptide hormone secreted by C-cells of The inherent capacity of many peptides to fibrillize and form thyroid gland. It is a hypocalcemic peptide involved in calcium home- amyloids evoked a lot of attention among researchers and realizes that ostasis and exerts its effects by antagonizing the effects of PTH [6]. some peptides and hormones can form protein supramolecular assem- Salmon calcitonin is preferred for clinical use because it is reported as blies of various forms and functionality [12,13]. Amyloids are now ⁎ Corresponding author at: Molecular Sciences Lab, National Institute of Immunology, Aruna Asaf Ali Marg, JNU Complex, New Delhi 110067, India. E-mail address: [email protected] (S. Gupta). https://doi.org/10.1016/j.bone.2019.02.019 Received 23 September 2018; Received in revised form 18 February 2019; Accepted 19 February 2019 Available online 21 February 2019 8756-3282/ © 2019 Elsevier Inc. All rights reserved. K. Manglani, et al. Bone 122 (2019) 123–135 considered as a new generic structure characterized by cross-beta-sheet dry for 2 min. After that each sample was rinsed with nanopure water formation, and this structure can be achieved by many peptides under and dried under a flow of nitrogen. Samples were imaged in air in non- suitable conditions [14]. During amyloid formation amorphous ag- contact acoustic AC mode using Atomic force microscope 5500 (Agilent gregates are generated as a prelude to gaining some new properties Technologies, USA). AC cantilever (normal spring constant of canti- such as enhanced stability, protease resistance, self-propagation, longer lever) was used. The topographic images of all samples were used for shelf life, highly organized structure which lacks soluble precursor analysis using PicoView software. protein and serve as a supramolecular structure made up of compact source of pure molecules [15]. 2.4. Fourier transform infrared spectroscopy Here, we hypothesized that whether the generic ability of human calcitonin to fibrillize could be utilized for formation of supramolecular IR spectra were recorded using Bruker Tensor 27 bench top FT-IR structures, a state that can harbor monomers in its native state. This spectrophotometer equipped with liquid nitrogen cooled mercury cad- property has been harnessed by our group previously to develop a mium telluride detector. SCA-I was analyzed on Bio-ATR and 256 in- sustained releasing supramolecular form of insulin and IL-1RA [16,17]. terferograms were recorded at room temperature with a resolution of − Herein we developed the supramolecular protein assembly of a hor- 2cm 1. mone whose pharmacological effect on the bone is still less studied. In this study we primarily describe the formation of supramolecular cal- 2.5. Proteinase K digestion assay citonin, its stability and ability to act as a sustained release form in vitro and in vivo. Apart from this, we employed supramolecular calcitonin Proteinase K digestion assay was performed as described previously assembly as a tool to evaluate the actual role of native human calcitonin [17]. SCA-I was incubated with Proteinase K (2 mg/ml; 1:1000 dilu- monomers on the bone and also to shed light on how human calcitonin tion) at 37 °C. Samples were drawn at 15 min and 30 min of incubation affects bone mass accrual. Thus, in present work we showed the char- and electrophoresed on 20% SDS-PAGE and visualized by Coomassie acterization of supramolecular calcitonin, its potency to reduce osteo- Brilliant Blue staining. clast activity and evaluated its effect on both trabecular and cortical bone compartments following a single injection in a rat ovariectomy 2.6. Cell viability assay model of bone loss. RAW 264.7 cells were seeded to 96 well microtiter plates with 2. Materials and methods starting density of 2.5 × 104 cells for each well. After 24 h cells were treated with PBS, mannitol (50 nM), human calcitonin (10 nM), SCA-I 2.1. Calcitonin fibril formation (25 μg) and dialysate (10 nM) of SCA-I for 24 h and 48 h. In each well, 10 μl of MTT was added from the stock (5 mg/ml) and incubated at For aggregation experiment, human/salmon calcitonin (AnaSpec, 37 °C in humidified CO2 incubator for 4 h. After incubation, medium USA) was used at a concentration of 2 mg/ml solubilized in 1× PBS was removed carefully and 100 μl of DMSO was added in each well to (pH 7.4) and incubated at 37 °C on thermal block for 100 h. No agita- dissolve the formazan crystals. After gentle shanking for 2 min the ab- tion is required for the aggregation of calcitonin. The kinetics of amy- sorbance was measured for each well at 560 nm using microplate reader loid fibril formation was monitored by measuring the turbidity of so- (Tecan Infinite M200) with background correction at 690 nm. % sur- lution at various time intervals using spectrophotometer (UV-2450 vival was calculated by [Absorbance(treatment) × 100] / Absorbance Shimadzu, Japan) at 600 nm and 340 nm for human and salmon cal- (control). citonin respectively. 2.7. Annexin V-FITC and PI staining and flow cytometry 2.2. SCA-I formation and in vitro release assay RAW 264.7 cells were plated in 24-well plate with starting density Human calcitonin (2 mg/ml each) in 1× PBS (pH 7.4) was in- of 5 × 104 and allowed to grow at 37 °C. After 24 h, cells were treated cubated at 37 °C for 4–6 h. When turbidity of solution reached between with PBS, mannitol (50 nM), human calcitonin (10 nM), SCA-I (25 μg) 0.8 and 1.0 OD at 600 nm, the reaction was stopped by keeping the and dialysate (10 nM) of SCA-I. After 48 h, cells were harvested with solution at 4 °C and the solution was centrifuged at 6000 rpm for 30 min trypsin EDTA and centrifuged at 800 rpm at 4 °C. After washing with ice at 4 °C. Protein concentration of supernatant (SCAeI) was measured cold PBS three times and cells were re-suspended in 100 μl Annexin V spectrophotometrically at 280 nm.