The existing suite of therapies for bone diseases largely act to prevent further bone loss but fail to stimulate healthy bone formation and repair. We describe an endogenous osteopeptide (PEPITEM) with anabolic osteogenic activity, regulating bone remodeling in health and disease. PEPITEM acts directly on osteoblasts through NCAM-1 signaling to promote their maturation and formation of new bone, leading to enhanced trabecular bone growth and strength. Simultaneously, PEPITEM stimulates an inhibitory paracrine loop: promoting osteoblast release of the decoy receptor osteoprotegerin, which sequesters RANKL, thereby limiting osteoclast activity and bone resorption. In disease models, PEPITEM therapy halts osteoporosis-induced bone loss and arthritis-induced bone damage in mice and stimulates new bone formation in osteoblasts derived from patient samples. Thus, PEPITEM offers an alternative therapeutic option in the management of diseases with excessive bone loss, promoting an endogenous anabolic pathway to induce bone remodeling and redress the imbalance in bone turnover.
Bone is a highly active organ, undergoing continuous osteoblast-induced bone formation and osteoclast-mediated bone resorption throughout life. The process of bone remodeling is orchestrated by cross-talk among osteoblasts, osteoclasts, and osteocytes acting in concert to maintain structural integrity, repair damage, and respond to changes in activity and load. Dysregulation of these pathways underpins numerous musculoskeletal (MSK) diseases where excessive bone resorption (e.g., osteoporosis, rheumatoid arthritis, periodontal disease, cancer-bone metastases) or abnormal bone formation (e.g., ankylosing spondylitis; heterotopic ossification) results in permanent loss of function, pain, increased risk of fracture, and frailty. Osteoporosis is the most common bone disease globally, affecting over 54 million individuals in the United States and accounting for 3 million broken bones at a cost of ∼$26 billion per annum. There are no cures for bone damage. Existing therapies have predominantly focused on slowing the rate of bone damage (e.g., bisphosphonates; denosumab, saracatinib), with only a handful of drugs able to promote bone repair currently approved (e.g., parathyroid hormone—PTH or romosozumab). Due to poor patient response, poor drug compliance, and drug-induced microfractures leading to atypical femur fractures, there is an urgent need to develop a new suite of therapies that lead to bone repair and regeneration in patients with MSK diseases to restore tissue homeostasis and functional integrity.
Approximately 10% of all bone in the human body is replaced annually, through a series of tightly coordinated sequential steps. Hematopoietic myeloid cells (monocytes) recruited to the bone differentiate into mononuclear osteoclast precursors in response to M-CSF (macrophage colony stimulating factor) and later RANKL (receptor activator of nuclear factor-κB ligand) stimulation, before fusing together to form multinucleated mature osteoclasts. Within osteoclast resorption pits, locally released chloride and hydrogen ions dissolve the bone mineral, while proteases (matrix metalloproteinase [MMP], cathepsin K) digest the collagen matrix resulting in bone resorption. Replacement of this resorbed bone is performed by osteoblasts, derived from mesenchymal stem cells (MSCs), that become committed to the osteoblast lineage upon activation of the transcriptional regulator Runx2. As osteoblast precursors start to differentiate, they express increased amounts of collagen (COL1A1) and matrix proteins, which are deposited as an unmineralized osteoid. Maturing osteoblasts subsequently release alkaline phosphatase (ALP) into the collagen-rich matrix resulting in the deposition of hydroxyapatite crystals, mineralization of the matrix, and formation of new bone.
Physiological bone remodeling is tightly regulated by the intricate cross-talk between osteoblasts and osteoclasts at the bone surface and via signaling from osteocytes embedded within the bone tissue itself. Osteoblasts and osteocytes release both positive (RANKL, M-CSF) and negative (osteoprotegerin [OPG]) regulators of osteoclastogenesis to control the rate of bone resorption. Conversely, osteoclasts and osteocytes produce anabolic stimuli that include sphingosine 1-phosphate, Wnt/β-catenin proteins, and bone morphogenic proteins that induce osteoblast precursor recruitment, differentiation, and survival. The highly conserved and ubiquitously expressed seven isoforms of the human 14-3-3 family act as adaptor proteins influencing the function of other proteins, and thus a multitude of signaling pathways, to regulate cellular responses. The name 14-3-3 derives from the elution fraction (14 th) and subsequent position (3.3) on starch electrophoresis gel when the family was first identified in brain tissue. Of particular relevance to bone homeostasis, two 14-3-3 family members have been reported to differentially regulate osteoblast function, with 14-3-3β significantly reducing and 14-3-3ξ enhancing osteoblastogenesis. We have previously identified a bioactive 14-amino acid peptide (PEPITEM) cleaved from 14-3-3ξ, which regulates the migration of monocytes (osteoclast precursors) into non-bone tissues during inflammation. Here we investigated the ability of PEPITEM to directly influence bone remodeling under homeostatic conditions and subsequently the therapeutic efficacy of PEPITEM in models of excessive bone loss.
Initially we examined whether PEPITEM intrinsically regulated bone remodeling under basal conditions over a 2-week period. PEPITEM therapy significantly increased bone volume (BV/TV), trabecular number, and thickness in both the tibia and vertebrae of adult mice, indicating that PEPITEM promotes bone formation. As expected, we observed a concomitant decrease in the gaps between individual trabeculae (trabecular separation), as the increased trabeculae created a denser, more interconnected trabecular network. Cortical bone turnover takes much longer than trabecular bone: ∼4–6 weeks. As expected, we observed no changes in the cortical bone following 2 weeks of PEPITEM treatment, but we did observe a significant increase in cortical bone parameters following 4 weeks of treatment. When comparing these findings with existing drugs targeting the bone, the effect size for PEPITEM on BV/TV at 2 weeks is comparable to that seen following treatment with the bisphosphonate zoledronic acid for 3 weeks or PTH for up to 4 weeks. Thus indicating PEPITEM is as efficient at inducing bone formation compared with current standard of care.
The enhanced trabecular parameters induced by PEPITEM translated to increased bone strength, with PEPITEM therapy significantly increasing the force at which bones fracture.
