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1.
Calcif Tissue Int ; 112(4): 452-462, 2023 04.
Article in English | MEDLINE | ID: mdl-36754901

ABSTRACT

Patients with type 2 diabetes mellitus (T2DM) experience a higher risk of fractures despite paradoxically exhibiting normal to high bone mineral density (BMD). This has drawn into question the applicability to T2DM of conventional fracture reduction treatments that aim to retain BMD. In a primary human osteoblast culture system, high glucose levels (25 mM) impaired cell proliferation and matrix mineralization compared to physiological glucose levels (5 mM). Treatment with parathyroid hormone (PTH, 10 nM), a bone anabolic agent, and cinacalcet (CN, 1 µM), a calcimimetic able to target the Ca2+-sensing receptor (CaSR), were tested for their effects on proliferation and differentiation. Strikingly, CN+PTH co-treatment was shown to promote cell growth and matrix mineralization under both physiological and high glucose conditions. CN+PTH reduced apoptosis by 0.9-fold/0.4-fold as measured by Caspase-3 activity assay, increased alkaline phosphatase (ALP) expression by 1.5-fold/twofold, increased the ratio of nuclear factor κ-B ligand (RANKL) to osteoprotegerin (OPG) by 2.1-fold/1.6-fold, and increased CaSR expression by 1.7-fold/4.6-fold (physiological glucose/high glucose). Collectively, these findings indicate a potential for CN+PTH combination therapy as a method to ameliorate the negative impact of chronic high blood glucose on bone remodeling.


Subject(s)
Diabetes Mellitus, Type 2 , Parathyroid Hormone , Humans , Cinacalcet/pharmacology , Cinacalcet/metabolism , Diabetes Mellitus, Type 2/metabolism , Osteoblasts/metabolism , Osteoprotegerin/metabolism , Glucose/metabolism , RANK Ligand/metabolism , Cells, Cultured
2.
J Child Orthop ; 13(5): 543-550, 2019 Oct 01.
Article in English | MEDLINE | ID: mdl-31695823

ABSTRACT

PURPOSE: Surgical interventions are routinely performed on children with osteogenesis imperfecta (OI) to stabilize long bones, often post fracture. We speculated that a combination of intramedullary reaming and intraosseous injection of recombinant bone morphogenetic protein-2 (BMP-2) could enhance periosteal ossification and ultimately cortical thickness and strength. This approach was conceptually tested in a preclinical model of genetic bone fragility. METHODS: Six experimental groups were tested including no treatment, intramedullary reaming, and reaming with 5 µg BMP-2 injection performed in the tibiae of both wild type (WT) and Col1a2 G610C/+ (OI, Amish mutation) mice. Bone formation was examined at a two-week time point in ex vivo specimens by micro-computed tomography (microCT) analysis and histomorphometry with a dynamic bone label. RESULTS: MicroCT data illustrated increases in tibial cortical thickness with intramedullary reaming alone (Saline) and reaming plus BMP-2 injection (BMP-2) compared to no intervention controls. In the OI mice, the periosteal bone increase was not statistically significant with Saline but there was an increase of +192% (p = 0.053) with BMP-2 injection. Dynamic histomorphometry on calcein label was used to quantify new woven bone formation; while BMP-2 induced greater bone formation than Saline, the anabolic response was blunted overall in the OI groups. CONCLUSIONS: These data indicate that targeting the intramedullary compartment via reaming and intraosseous BMP-2 delivery can lead to gains in cortical bone parameters. It is suggested that the next step is to validate safety and functional improvements in a clinical OI setting.

3.
Calcif Tissue Int ; 103(5): 529-539, 2018 11.
Article in English | MEDLINE | ID: mdl-29943187

ABSTRACT

Significant fracture history in children is defined as having at least one vertebral fracture, at least 2 fractures by age 10, or at least 3 fractures by age 19. Between September 2011 and December 2014, clinical data were collected on children with a significant fracture history that attended a major Australian children's hospital. Fifty-six patients were identified as having 305 fractures in total, including 44 vertebral fractures. 18% of patients (10/56) were diagnosed with osteogenesis imperfecta (OI) by a bone health expert, molecular testing or both, and they sustained 23% of all fractures (71/305). Analysis of serum bone biochemistry showed all median values to be within a normal range and no clinically significant differences between patients with and without OI. The DXA and pQCT derived bone mineral density (BMD) and bone mineral content (BMC) Z scores were reduced overall. DXA derived total body and lumbar spine areal BMD-for-age and BMC-for-age Z scores were significantly lower in children who had vertebral fractures or who were later diagnosed with OI. Similarly, pQCT performed on radii and tibiae showed Z scores significantly less than zero. pQCT-derived limb muscle cross sectional area Z scores were significantly lower in the OI subgroup. In conclusion, this study describes the bone phenotype of children referred to a tertiary hospital clinic for recurrent fractures and highlights a subset of children with previously undiagnosed OI, but a larger cohort without classic OI. Thus it can be clinically challenging to differentiate between children with OI type 1 (mild phenotype) and non-OI children without bone densitometry and genetic testing. We conclude that recurrent fractures in children should prompt a comprehensive bone and systemic health assessment to eliminate an underlying pathology.


Subject(s)
Fractures, Bone/diagnosis , Fractures, Bone/etiology , Adolescent , Child , Child, Preschool , Cohort Studies , Female , Humans , Infant , Male , Osteogenesis Imperfecta/complications , Recurrence
4.
J Musculoskelet Neuronal Interact ; 15(2): 161-70, 2015 Jun.
Article in English | MEDLINE | ID: mdl-26032208

ABSTRACT

Neurofibromatosis Type 1 (NF1) is a genetic neurocutaneous disorder with multisystem manifestations, including a predisposition to tumor formation and bone dysplasias. Studies over the last decade have shown that NF1 can also be associated with significant motor deficits, such as poor coordination, low muscle tone, and easy fatigability. These have traditionally been ascribed to developmental central nervous system and cognitive deficits. However, recent preclinical studies have also illustrated a primary role for the NF1 gene product in muscle growth and metabolism; these findings are consistent with clinical studies demonstrating reduced muscle size and muscle weakness in individuals with NF1. Currently there is no evidence-based intervention for NF1 muscle and motor deficiencies; this review identifies key research areas where improved mechanistic understanding could unlock new therapeutic options.


Subject(s)
Movement Disorders/physiopathology , Muscle, Skeletal/pathology , Neurofibromatosis 1/pathology , Adolescent , Adult , Child , Evidence-Based Medicine , Female , Humans , Male , Muscle, Skeletal/physiopathology , Neurofibromatosis 1/physiopathology , Neurofibromatosis 1/therapy , Young Adult
5.
J Bone Joint Surg Am ; 96(14): e117, 2014 Jul 16.
Article in English | MEDLINE | ID: mdl-25031379

ABSTRACT

BACKGROUND: Congenital tibial dysplasia is a severe pediatric condition that classically results in a persistent pseudarthrosis. A majority of these cases are associated with neurofibromatosis type I (NF1), a genetic disorder in which inactivation of the NF1 gene leads to overactivity of the Ras-MEK-MAPK (mitogen-activated protein kinase) signaling pathway. We therefore hypothesized that pharmaceutical inhibition of MEK-MAPK may be a beneficial therapeutic strategy. METHODS: In vitro methods were used to demonstrate a role for the MEK inhibitor PD0325901 in promoting osteogenic differentiation in Nf1-/- calvarial osteoblasts. Local applications of rhBMP-2 and/or PD0325901 were then tested in a mouse model of NF1 tibial pseudarthrosis featuring localized double inactivation of the Nf1 gene in a fracture. Mice received no treatment, PD0325901 (10 mg/kg/day from two days before fracture to ten days after fracture), rhBMP-2 (10 µg), or a combination of rhBMP-2 and PD0325901. RESULTS: Animals treated with the delivery vehicle alone, PD0325901, rhBMP-2, or the PD0325901 + rhBMP-2 combination showed union rates of 0%, 8%, 69% (p < 0.01), or 80% (p < 0.01), respectively, at twenty-one days after fracture. Mice treated with the rhBMP-2 + PD0325901 combination displayed a callus volume sixfold greater than the vehicle controls and twofold greater than the group receiving rhBMP-2 alone. Although MEK inhibition combined with rhBMP-2 led to increases in bone formation and union, the proportion of fibrous tissue in the callus was not significantly reduced. CONCLUSIONS: The data suggest that MEK inhibition can promote bone formation in combination with rhBMP-2 in the context of an NF1 pseudarthrosis. However, PD0325901 did not promote substantive bone anabolism in the absence of an exogenous anabolic stimulus and did not suppress fibrosis. CLINICAL RELEVANCE: This study examines a signaling pathway-based approach to treating poor bone healing in a model of NF1 pseudarthrosis.


Subject(s)
Benzamides/administration & dosage , Bone Morphogenetic Protein 2/administration & dosage , Diphenylamine/analogs & derivatives , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Neurofibromatosis 1/complications , Osteogenesis/drug effects , Pseudarthrosis/drug therapy , Pseudarthrosis/etiology , Transforming Growth Factor beta/administration & dosage , Animals , Benzamides/pharmacology , Bone Morphogenetic Protein 2/pharmacology , Diphenylamine/administration & dosage , Diphenylamine/pharmacology , Disease Models, Animal , Drug Therapy, Combination , Female , Mice , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacology , Transforming Growth Factor beta/pharmacology
6.
J Bone Miner Res ; 29(11): 2456-67, 2014 Nov.
Article in English | MEDLINE | ID: mdl-24821585

ABSTRACT

Sclerostin, encoded by the Sost gene, is an important negative regulator of bone formation that has been proposed to have a key role in regulating the response to mechanical loading. To investigate the effect of long-term Sclerostin deficiency on mechanotransduction in bone, we performed experiments on unloaded or loaded tibiae of 10 week old female Sost-/- and wild type mice. Unloading was induced via 0.5U botulinum toxin (BTX) injections into the right quadriceps and calf muscles, causing muscle paralysis and limb disuse. On a separate group of mice, increased loading was performed on the left tibiae through unilateral cyclic axial compression of equivalent strains (+1200 µe) at 1200 cycles/day, 5 days/week. Another cohort of mice receiving equivalent loads (-9.0 N) also were assessed. Contralateral tibiae served as normal load controls. Loaded/unloaded and normal load tibiae were assessed at day 14 for bone volume (BV) and formation changes. Loss of BV was seen in the unloaded tibiae of wild type mice, but BV was not different between normal load and unloaded Sost-/- tibiae. An increase in BV was seen in the loaded tibiae of wild type and Sost-/- mice over their normal load controls. The increased BV was associated with significantly increased mid-shaft periosteal mineralizing surface/bone surface (MS/BS), mineral apposition rate (MAR), and bone formation rate/bone surface (BFR/BS), and endosteal MAR and BFR/BS. Notably, loading induced a greater increase in periosteal MAR and BFR/BS in Sost-/- mice than in wild type controls. Thus, long-term Sclerostin deficiency inhibits the bone loss normally induced with decreased mechanical load, but it can augment the increase in bone formation with increased load.


Subject(s)
Calcification, Physiologic/physiology , Glycoproteins/deficiency , Mechanotransduction, Cellular/physiology , Osteogenesis/physiology , Periosteum/metabolism , Tibia/metabolism , Adaptor Proteins, Signal Transducing , Animals , Botulinum Toxins/toxicity , Calcification, Physiologic/drug effects , Female , Intercellular Signaling Peptides and Proteins , Mechanotransduction, Cellular/drug effects , Mice , Mice, Knockout , Osteogenesis/drug effects , Paralysis/chemically induced , Paralysis/genetics , Paralysis/metabolism , Weight-Bearing
7.
Eur Cell Mater ; 27: 98-109; discussion 109-111, 2014 Jan 31.
Article in English | MEDLINE | ID: mdl-24488823

ABSTRACT

Current clinical delivery of recombinant human bone morphogenetic proteins (rhBMPs) utilises freeze-dried collagen. Despite effective new bone generation, rhBMP via collagen can be limited by significant complications due to inflammation and uncontrolled bone formation. This study aimed to produce an alternative rhBMP local delivery system to permit more controllable and superior rhBMP-induced bone formation. Cylindrical porous poly(lactic-co-glycolic acid) (PLGA) scaffolds were manufactured by thermally-induced phase separation. Scaffolds were encapsulated with anabolic rhBMP-2 (20 µg) ± anti-resorptive agents: zoledronic acid (5 µg ZA), ZA pre-adsorbed onto hydroxyapatite microparticles, (5 µg ZA/2% HA) or IkappaB kinase (IKK) inhibitor (10 µg PS-1145). Scaffolds were inserted in a 6-mm critical-sized femoral defect in Wistar rats, and compared against rhBMP-2 via collagen. The regenerate region was examined at 6 weeks by 3D microCT and descriptive histology. MicroCT and histology revealed rhBMP-induced bone was more restricted in the PLGA scaffolds than collagen scaffolds (-92.3% TV, p < 0.01). The regenerate formed by PLGA + rhBMP-2/ZA/HA showed comparable bone volume to rhBMP-2 via collagen, and bone mineral density was +9.1% higher (p < 0.01). Local adjunct ZA/HA or PS-1145 significantly enhanced PLGA + rhBMP-induced bone formation by +78.2% and +52.0%, respectively (p ≤ 0.01). Mechanistically, MG-63 human osteoblast-like cells showed cellular invasion and proliferation within PLGA scaffolds. In conclusion, PLGA scaffolds enabled superior spatial control of rhBMP-induced bone formation over clinically-used collagen. The PLGA scaffold has the potential to avoid uncontrollable bone formation-related safety issues and to customise bone shape by scaffold design. Moreover, local treatment with anti-resorptive agents incorporated within the scaffold further augmented rhBMP-induced bone formation.


Subject(s)
Bone Density Conservation Agents/pharmacology , Bone Morphogenetic Protein 2/metabolism , Bone Regeneration , Diphosphonates/pharmacology , Guided Tissue Regeneration , Imidazoles/pharmacology , Tissue Scaffolds/chemistry , Animals , Bone Morphogenetic Protein 2/genetics , Cell Line , Femur/drug effects , Femur/surgery , Humans , I-kappa B Kinase/antagonists & inhibitors , Lactic Acid/pharmacology , Male , Osteoblasts/drug effects , Osteoblasts/metabolism , Polyglycolic Acid/pharmacology , Polylactic Acid-Polyglycolic Acid Copolymer , Rats , Rats, Wistar , Zoledronic Acid
8.
Bone ; 59: 151-61, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24269278

ABSTRACT

MEK inhibitors (MEKi) PD0325901 and AZD6244 (Selumetinib) are drugs currently under clinical investigation for cancer treatment, however the Ras-MAPK pathway is also an important mediator of normal bone cell differentiation and function. In this study we examined the effects of these compounds on endochondral processes using both in vitro and in vivo models. Treatment with PD0325901 or AZD6244 significantly increased Runx2 and Alkaline phosphate gene expression in calvarial osteoblasts and decreased TRAP+ cells in induced osteoclast cultures. To test the effects of these drugs on bone healing, C57/Bl6 mice underwent a closed tibial fracture and were treated with PD0325901 or AZD6244 at 10mg/kg/day. Animals were culled at day 10 and at day 21 post-fracture for analysis of the fracture callus and the femoral growth plate in the contralateral leg. MEKi treatment markedly increased cartilage volume in the soft callus at day 10 post-fracture (+60% PD0325901, +20% AZD6244) and continued treatment led to a delay in cartilage remodeling. At the growth plate, we observed an increase in the height of the hypertrophic zone relative to the proliferative zone of +78% in PD0325901 treated mice. Osteoclast surface was significantly decreased both at the terminal end of the growth plate and within the fracture calluses of MEKi treated animals. The mechanistic effects of MEKi on genes encoding cartilage matrix proteins and catabolic enzymes were examined in articular chondrocyte cultures. PD0325901 or AZD6244 led to increased matrix protein expression (Col2a1 and Acan) and decreased expression of catabolic factors (Mmp13 and Adamts-5). Taken together, these data support the hypothesis that MEKi treatment can impact chondrocyte hypertrophy, matrix resorption, and fracture healing. These compounds can also affect bone architecture by expanding the hypertrophic zone of the growth plate and reducing osteoclast surface systemically.


Subject(s)
Benzamides/pharmacology , Benzimidazoles/pharmacology , Diphenylamine/analogs & derivatives , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Osteogenesis/drug effects , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Bony Callus/drug effects , Bony Callus/pathology , Cartilage/drug effects , Cartilage/growth & development , Cell Differentiation/drug effects , Chondrocytes/drug effects , Chondrocytes/metabolism , Chondrocytes/pathology , Diphenylamine/pharmacology , Fracture Healing/drug effects , MAP Kinase Signaling System/drug effects , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase Kinases/metabolism , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoblasts/enzymology , Osteoclasts/drug effects , Osteoclasts/enzymology , Osteoprotegerin/blood , Protein Kinase Inhibitors/pharmacology , RANK Ligand/blood , Sheep , Skull/cytology , X-Ray Microtomography
9.
Eur Cell Mater ; 26: 208-21; discussion 220-1, 2013 Oct 22.
Article in English | MEDLINE | ID: mdl-24146213

ABSTRACT

Bone tissue engineering approaches commonly involve the delivery of recombinant human bone morphogenetic proteins (rhBMPs). However, there are limitations associated with the currently used carriers, including the need for surgical implantation and the associated increase in infection risk. As an alternative to traditional porous collagen sponge, we have adopted a solution of the injectable sucrose acetate isobutyrate (SAIB) as a carrier for rhBMP-2. The ability to deliver rhBMP-2 and other agents by injection reduces the infection risk and lesion size whilst in surgery, with the potential to avoid open surgery altogether in some indications. The primary methodology used for this in vivo study was a C57BL6/J mouse ectopic bone formation model. Specimens were examined by x-ray, microCT, and histology at 3 weeks. SAIB was delivered non-invasively and produced up to 3-fold greater bone volume compared to collagen. To further refine and improve upon the formulation, SAIB containing rhBMP-2 was admixed with candidate compounds including ceramic microparticles, anti-resorptives, and cell signalling inhibitors and further tested in vivo. The formulation combining SAIB/rhBMP-2, the bisphosphonate zoledronic acid (ZA), and hydroxyapatite (HA) microparticles yielded a 10-fold greater bone volume than SAIB/rhBMP-2 alone. To investigate the mechanism underlying the synergy between ZA and HA, we used in vitro binding assays and in vivo fluorescent biodistribution studies to demonstrate that ceramic particles could bind and sequester the bisphosphonate. These data show the utility of SAIB as a non-invasive rhBMP delivery system as well as describing an optimised formulation for bone tissue engineering.


Subject(s)
Bone Regeneration , Drug Delivery Systems/methods , Sucrose/analogs & derivatives , Tissue Engineering , Animals , Bone Morphogenetic Protein 2/administration & dosage , Cell Culture Techniques , Collagen/therapeutic use , Diphosphonates/pharmacokinetics , Diphosphonates/therapeutic use , Hydroxyapatites/pharmacokinetics , Hydroxyapatites/therapeutic use , Imidazoles/pharmacokinetics , Imidazoles/therapeutic use , Mice , Mice, Inbred C57BL , Sucrose/pharmacokinetics , Sucrose/therapeutic use , Tissue Distribution , Zoledronic Acid
10.
J Bone Joint Surg Br ; 93(8): 1134-9, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21768643

ABSTRACT

Congenital pseudarthrosis of the tibia is an uncommon manifestation of neurofibromatosis type 1 (NF1), but one that remains difficult to treat due to anabolic deficiency and catabolic excess. Bone grafting and more recently recombinant human bone morphogenetic proteins (rhBMPs) have been identified as pro-anabolic stimuli with the potential to improve the outcome after surgery. As an additional pharmaceutical intervention, we describe the combined use of rhBMP-2 and the bisphosphonate zoledronic acid in a mouse model of NF1-deficient fracture repair. Fractures were generated in the distal tibiae of neurofibromatosis type 1-deficient (Nf1(+/-)) mice and control mice. Fractures were open and featured periosteal stripping. All mice received 10 µg rhBMP-2 delivered in a carboxymethylcellulose carrier around the fracture as an anabolic stimulus. Bisphosphonate-treated mice also received five doses of 0.02 mg/kg zoledronic acid given by intraperitoneal injection. When only rhBMP but no zoledronic acid was used to promote repair, 75% of fractures in Nf1(+/-) mice remained ununited at three weeks compared with 7% of controls (p < 0.001). Systemic post-operative administration of zoledronic acid halved the rate of ununited fractures to 37.5% (p < 0.07). These data support the concept that preventing bone loss in combination with anabolic stimulation may improve the outcome following surgical treatment for children with congenital pseudarthosis of the tibia and NF1.


Subject(s)
Bone Density Conservation Agents/therapeutic use , Bone Morphogenetic Protein 2/therapeutic use , Diphosphonates/therapeutic use , Imidazoles/therapeutic use , Neurofibromatosis 1/complications , Tibial Fractures/drug therapy , Animals , Combined Modality Therapy , Disease Models, Animal , Drug Evaluation, Preclinical/methods , Drug Therapy, Combination , Fracture Fixation, Internal/methods , Fracture Healing/drug effects , Fractures, Ununited/prevention & control , Mice , Mice, Knockout , Postoperative Care/methods , Pseudarthrosis/complications , Pseudarthrosis/congenital , Recombinant Proteins/therapeutic use , Tibial Fractures/diagnostic imaging , Tibial Fractures/etiology , Tibial Fractures/surgery , Treatment Outcome , X-Ray Microtomography , Zoledronic Acid
11.
J Musculoskelet Neuronal Interact ; 10(1): 71-6, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20190382

ABSTRACT

Bone repair is a complex phenomenon involving many cell types and signaling factors. Substantial evidence exists to suggest that stem cells originating from local osseous tissues, particularly the periosteum, can contribute to bone repair. However, there are situations where injury or post-surgical management can deplete the amount of, and/or access to these crucial progenitors. The fact that bone repair can still occur in these circumstances implicitly reflects the existence of compensatory secondary systems. One potential alternate source of osteoprogenitors is muscle, which is closely associated with bone and typically suffers trauma during an orthopedic insult. While muscle access is known to be beneficial to bone repair, this is conventionally credited to its high vascularity, and thus its contribution to the local blood supply. However, there is emerging evidence to suggest that progenitors from muscle may directly contribute to bone healing. Defining the role of muscle in bone formation and repair has significant clinical implications, particularly where promoting access to this tissue may enhance the repair outcome.


Subject(s)
Bone Regeneration/physiology , Bone and Bones/metabolism , Muscle, Skeletal/metabolism , Osteogenesis/physiology , Animals , Bone and Bones/cytology , Fractures, Bone/metabolism , Fractures, Bone/physiopathology , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/cytology , Periosteum/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Wound Healing/physiology
12.
J Bone Joint Surg Br ; 89(4): 425-33, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17463107

ABSTRACT

The literature on fracture repair has been reviewed. The traditional concepts of delayed and nonunion have been examined in terms of the phased and balanced anabolic and catabolic responses in bone repair. The role of medical manipulation of these inter-related responses in the fracture healing have been considered.


Subject(s)
Fracture Healing/physiology , Fractures, Bone/metabolism , Anabolic Agents/therapeutic use , Bone Morphogenetic Proteins/therapeutic use , Fractures, Ununited/drug therapy , Fractures, Ununited/metabolism , Fractures, Ununited/physiopathology , Humans , Models, Biological
14.
J Cell Biol ; 153(5): 985-98, 2001 May 28.
Article in English | MEDLINE | ID: mdl-11381084

ABSTRACT

We have isolated a murine cDNA encoding a 9-kD protein, Chisel (Csl), in a screen for transcriptional targets of the cardiac homeodomain factor Nkx2-5. Csl transcripts were detected in atria and ventricles of the heart and in all skeletal muscles and smooth muscles of the stomach and pulmonary veins. Csl protein was distributed throughout the cytoplasm in fetal muscles, although costameric and M-line localization to the muscle cytoskeleton became obvious after further maturation. Targeted disruption of Csl showed no overt muscle phenotype. However, ectopic expression in C2C12 myoblasts induced formation of lamellipodia in which Csl protein became tethered to membrane ruffles. Migration of these cells was retarded in a monolayer wound repair assay. Csl-expressing myoblasts differentiated and fused normally, although in the presence of insulin-like growth factor (IGF)-1 they showed dramatically enhanced fusion, leading to formation of large dysmorphogenic "myosacs." The activities of transcription factors nuclear factor of activated T cells (NFAT) and myocyte enhancer-binding factor (MEF)2, were also enhanced in an IGF-1 signaling-dependent manner. The dynamic cytoskeletal localization of Csl and its dominant effects on cell shape and behavior and transcription factor activity suggest that Csl plays a role in the regulatory network through which muscle cells coordinate their structural and functional states during growth, adaptation, and repair.


Subject(s)
Insulin-Like Growth Factor I/pharmacology , Muscle Proteins/metabolism , Muscles/cytology , Muscles/drug effects , Nuclear Proteins , Xenopus Proteins , Aging/metabolism , Amino Acid Sequence , Animals , Base Sequence , Calcineurin/metabolism , Cell Differentiation , Cell Fusion , Cell Line , Cell Size/drug effects , Cytoskeleton/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation/drug effects , Homeobox Protein Nkx-2.5 , Homeodomain Proteins/metabolism , MEF2 Transcription Factors , Mice , Mice, Knockout , Molecular Sequence Data , Muscle Proteins/chemistry , Muscle Proteins/genetics , Muscles/embryology , Muscles/metabolism , Myogenic Regulatory Factors , NFATC Transcription Factors , Organ Specificity , Physical Chromosome Mapping , Protein Transport , RNA, Messenger/analysis , RNA, Messenger/genetics , Transcription Factors/metabolism , Wound Healing
15.
Biochemistry ; 38(25): 7978-88, 1999 Jun 22.
Article in English | MEDLINE | ID: mdl-10387041

ABSTRACT

High-resolution crystal structures are described for seven macrocycles complexed with HIV-1 protease (HIVPR). The macrocycles possess two amides and an aromatic group within 15-17 membered rings designed to replace N- or C-terminal tripeptides from peptidic inhibitors of HIVPR. Appended to each macrocycle is a transition state isostere and either an acyclic peptide, nonpeptide, or another macrocycle. These cyclic analogues are potent inhibitors of HIVPR, and the crystal structures show them to be structural mimics of acyclic peptides, binding in the active site of HIVPR via the same interactions. Each macrocycle is restrained to adopt a beta-strand conformation which is preorganized for protease binding. An unusual feature of the binding of C-terminal macrocyclic inhibitors is the interaction between a positively charged secondary amine and a catalytic aspartate of HIVPR. A bicyclic inhibitor binds similarly through its secondary amine that lies between its component N-terminal and C-terminal macrocycles. In contrast, the corresponding tertiary amine of the N-terminal macrocycles does not interact with the catalytic aspartates. The amine-aspartate interaction induces a 1.5 A N-terminal translation of the inhibitors in the active site and is accompanied by weakened interactions with a water molecule that bridges the ligand to the enzyme, as well as static disorder in enzyme flap residues. This flexibility may facilitate peptide cleavage and product dissociation during catalysis. Proteases [Aba67,95]HIVPR and [Lys7,Ile33,Aba67,95]HIVPR used in this work were shown to have very similar crystal structures.


Subject(s)
HIV Protease Inhibitors/chemistry , HIV-1/enzymology , Molecular Mimicry , Peptides, Cyclic/chemistry , Aminobutyrates/chemistry , Asparagine/chemistry , Binding Sites , Computer Simulation , Crystallography, X-Ray , HIV Protease Inhibitors/chemical synthesis , Isoleucine/chemistry , Leucine/chemistry , Lysine/chemistry , Models, Molecular , Oligopeptides/chemistry , Peptides, Cyclic/chemical synthesis , Phenylalanine/chemistry , Protein Conformation , Stereoisomerism , Valine/chemistry
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