Bone biology: insights from osteogenesis imperfecta and related rare fragility syndromes

Abstract

The limited accessibility of bone and its mineralized nature have restricted deep investigation of its biology. Recent breakthroughs in identification of mutant proteins affecting bone tissue homeostasis in rare skeletal diseases have revealed novel pathways involved in skeletal development and maintenance. The characterization of new dominant, recessive and X-linked forms of the rare brittle bone disease osteogenesis imperfecta (OI) and other OI-related bone fragility disorders was a key player in this advance. The development of in vitro models for these diseases along with the generation and characterization of murine and zebrafish models contributed to dissecting previously unknown pathways. Here, we describe the most recent advances in the understanding of processes involved in abnormal bone mineralization, collagen processing and osteoblast function, as illustrated by the characterization of new causative genes for OI and OI-related fragility syndromes. The coordinated role of the integral membrane protein BRIL and of the secreted protein PEDF in modulating bone mineralization as well as the function and cross-talk of the collagen-specific chaperones HSP47 and FKBP65 in collagen processing and secretion are discussed. We address the significance of WNT ligand, the importance of maintaining endoplasmic reticulum membrane potential and of regulating intramembrane proteolysis in osteoblast homeostasis. Moreover, we also examine the relevance of the cytoskeletal protein plastin-3 and of the nucleotidyltransferase FAM46A. Thanks to these advances, new targets for the development of novel therapies for currently incurable rare bone diseases have been and, likely, will be identified, supporting the important role of basic science for translational approaches.

Keywords: ER Golgi trafficking; bone biology; bone mineralization; collagen; intramembrane proteolysis; nucleotidyltransferase; osteoblast differentiation; osteogenesis imperfecta; plastin 3; skeletal signaling pathways.

Figure 1:. BRIL and PEDF regulation of bone mineralization.

The bone restricted ifitm-like protein (BRIL) on osteoblasts membrane regulates the expression of the SERPINF1 gene, encoding for pigment epithelium derived factor (PEDF). In presence of the dominant c.−14C˃T mutation in the 5′-untranslated region of the interferon-induced transmembrane protein 5 (IFITM5) gene, a novel in-frame translation start site is created that adds 5 amino acid (MALEP) at the cytosolic N-terminus of BRIL. MALEP-BRIL increases the expression of PEDF. In presence of the heterozygous p.Ser40Leu substitution in BRIL, palmitoylation is impaired and the mutant protein is retained in the Golgi, being unable to regulate gene expression. Dotted lines refer to the presence of still unknown factors in the pathway. On osteocyte membrane PEDF binds to PEDF-R inducing phosphorylation of ERK. Active ERK phosphorylates glycogen synthase kinase 3-beta (GSK-3β), targeting it for destruction and leading to the stabilization of β-catenin. β-catenin migrates into the nucleus inducing transcription of the target genes.

Figure 2:. HSP47 role in type I collagen triple helix stabilization and secretion.

Heat shock protein 47 (HSP47) is an ER resident chaperone able to specifically bind type I procollagen molecules, preventing local unfolding and lateral aggregation and/or fibril formation. In the ER, HSP47 interacts with the chaperone peptidyl prolyl cis-trans isomerase 65-KDa FK506-binding protein (FKBP65) and the lysyl hydroxylase 2 (LH2). The stability of this complex is regulated by the master effector of the unfolded protein response (UPR) pathway, Bip. Upon binding to HSP47, procollagen trafficking towards Golgi is mediated by the formation of larger COPII vesicles derived by the HSP47 anchorage to the SH3 domain of the ER protein TANGO1. Once reaching the Golgi, HSP47 releases its cargo in a pH dependent manner and it is recycled back to the ER thanks to the presence of a C-terminal RDEL retention signal. HSP47 interacts also with the Ire1α effector of the UPR, thus modulating the adaptive UPR.

Figure 3:. TRIC-B, SP2, OASIS and SPARC function in bone homeostasis.

Collagen is synthesized within the endoplasmic reticulum (ER), the major intracellular Ca²⁺ store. In response to extracellular stimuli, Ca²⁺ is released from the ER lumen into the cytoplasm mainly via inositol-3-phosphate receptor (IP₃R) and it is transported back into the ER via SERCA pumps. The trimeric intracellular cation channel subtype B (TRIC-B) regulates Ca²⁺ fluctuations indirectly mediating transmembrane K⁺ flux to maintain electroneutrality across the ER membrane. In the absence of TRIC-mediated K⁺ flux, ER-resident Ca²⁺-binding chaperones, including BiP, Cyclophilin B (CyPB) and protein disulfide isomerase (PDI), that directly interact with collagen chains for assembly and folding, are dysregulated. Thus, absence of TRIC-B affects synthesis and secretion of collagen. S2P is a protease in the Golgi membrane involved in the Regulated Intramembrane Proteolysis (RIP) of transcription factors, such as old astrocyte specifically induced substance (OASIS). Following ER stress or sterol deficiency, OASIS is transported from the ER to Golgi membrane for sequential processing by S2P. SPARC is a calcium-binding protein, one of the most abundant non-collagenous protein expressed in mineralized tissues and relevant for cellular-matrix interaction. In osteoid, SPARC has been proposed to bind collagen and hydroxyapatite crystals and release calcium ions, perhaps enhancing mineralization of the bone collagen matrix. SPARC seems to have also an intracellular chaperone role since dermal fibroblasts isolated from OI individuals with SPARC mutations exhibited delay in collagen folding.

Figure 4:. WNT1 and FAM46A regulate osteoblast gene transcription.

Wingless-type MMTV integration site family 1 (WNT1) regulates bone formation in several ways. In the canonical pathway, WNT1 binding to a membrane dual receptor complex LRP5/6-Frizzle favors β-catenin release from a degradation complex and its migration to the nucleus where it regulates the expression of genes involved in osteoblasts differentiation and activity [109]. Recently, a WNT1 stimulatory effect on bone formation, independent from LRP5 and mediated by WNT1 regulation on the mammalian target of rapamycin (mTOR) receptor, has been hypothesized (gray shade). The WNT1/mTORC1 complex activates protein synthesis through the phosphorylation of the ribosomal protein S6. Question mark refer to the presence of still unknown factors in the pathway. Following bone morphogenetic protein (BMP) association with the BMP receptor (BMPR) on osteoblast membrane, FAM46A binds to the receptor regulated SMAD (R-SMAD), stabilizing it and avoiding its degradation. Then, FAM46A, R-SMAD, and Co-SMAD form a complex that migrates to the nucleus and regulates gene expression activating, among others, the transcription of the BMP target genes [183].

Figure 5:. PLS3 function in osteoclasts.

Plastin 3 (PLS3) is a cytoskeletal protein involved in the formation of F-actin bundles. Osteoclast resorptive activity, migration and adhesion are dependent on formation of large actin filaments containing ring structures known as podosomes, which are impaired when PLS3 levels are decreased. In osteoclasts PLS3 interacts in the cytosol with the NF-kB repressing Factor (NKRF) favoring its translocation into the nucleus. Here NKRF inhibits the transcription of NFATC1, a key regulator for osteoclastogenesis, causing an impairment in osteoclast activity.