Objectives
Skeletal malocclusions are common, and severe malocclusions are treated by invasive surgeries. Recently, jaw bone length has been shown to be developmentally controlled by osteoclasts. Our ...objective was to determine the effect of inhibiting osteoclast‐secreted proteolytic enzymes on lower jaw bone length of avian embryos by pharmacologically inhibiting matrix metalloproteinase‐9 (MMP9) or cathepsin K (CTSK).
Methods
Quail (Coturnix coturnix japonica) embryos were given a single dose of an inhibitor of MMP9 (iMMP9), an inhibitor CTSK (iCTSK), or vehicle at a developmental stage when bone deposition is beginning to occur. At a developmental stage when the viscerocranium is largely calcified, the heads were scanned via micro‐computed tomography and reproducible landmarks were placed on 3D‐reconstructed skulls; the landmark coordinates were used to quantify facial bone dimensions.
Results
Approximately half of the quail given either iMMP9 or iCTSK demonstrated an overt lower jaw phenotype, characterized by longer lower jaw bones and a greater lower to upper jaw ratio than control embryos. Additionally, iMMP9‐treated embryos exhibited a significant change in midface length and iCTSK‐treated embryos had significant change in nasal bone length.
Conclusion
MMP9 and CTSK play a role in osteoclast‐mediated determination of lower jaw bone length. Pharmacological inhibition of MMP9 or CTSK may be a promising therapeutic alternative to surgery for treating skeletal jaw malocclusions, but more preclinical research is needed prior to clinical translation.
CCN2/Connective Tissue Growth Factor (CTGF) is a matricellular protein that regulates cell adhesion, migration, and survival. CCN2 is best known for its ability to promote fibrosis by mediating the ...ability of transforming growth factor β (TGFβ) to induce excess extracellular matrix production. In addition to its role in pathological processes, CCN2 is required for chondrogenesis. CCN2 is also highly expressed during development in endothelial cells, suggesting a role in angiogenesis. The potential role of CCN2 in angiogenesis is unclear, however, as both pro- and anti-angiogenic effects have been reported. Here, through analysis of Ccn2-deficient mice, we show that CCN2 is required for stable association and retention of pericytes by endothelial cells. PDGF signaling and the establishment of the endothelial basement membrane are required for pericytes recruitment and retention. CCN2 induced PDGF-B expression in endothelial cells, and potentiated PDGF-B-mediated Akt signaling in mural (vascular smooth muscle/pericyte) cells. In addition, CCN2 induced the production of endothelial basement membrane components in vitro, and was required for their expression in vivo. Overall, these results highlight CCN2 as an essential mediator of vascular remodeling by regulating endothelial-pericyte interactions. Although most studies of CCN2 function have focused on effects of CCN2 overexpression on the interstitial extracellular matrix, the results presented here show that CCN2 is required for the normal production of vascular basement membranes.
CCN2 (connective tissue growth factor (CTGF/CCN2)) is a matricellular protein that utilizes integrins to regulate cell proliferation, migration and survival. The loss of CCN2 leads to perinatal ...lethality resulting from a severe chondrodysplasia. Upon closer inspection of
Ccn2
mutant mice, we observed defects in extracellular matrix (ECM) organization and hypothesized that the severe chondrodysplasia caused by loss of CCN2 might be associated with defective chondrocyte survival.
Ccn2
mutant growth plate chondrocytes exhibited enlarged endoplasmic reticula (ER), suggesting cellular stress. Immunofluorescence analysis confirmed elevated stress in
Ccn2
mutants, with reduced stress observed in
Ccn2
overexpressing transgenic mice. In vitro studies revealed that
Ccn2
is a stress responsive gene in chondrocytes. The elevated stress observed in
Ccn2
−/− chondrocytes is direct and mediated in part through integrin α5. The expression of the survival marker NFκB and components of the autophagy pathway were decreased in
Ccn2
mutant growth plates, suggesting that CCN2 may be involved in mediating chondrocyte survival. These data demonstrate that absence of a matricellular protein can result in increased cellular stress and highlight a novel protective role for CCN2 in chondrocyte survival. The severe chondrodysplasia caused by the loss of CCN2 may be due to increased chondrocyte stress and defective activation of autophagy pathways, leading to decreased cellular survival. These effects may be mediated through nuclear factor κB (NFκB) as part of a CCN2/integrin/NFκB signaling cascade.
Normal hearing requires exquisite cooperation between bony and sensorineural structures within the cochlea. For example, the inner ear secretes proteins such as osteoprotegrin (OPG) that can prevent ...cochlear bone remodeling. Accordingly, diseases that affect bone regulation can also result in hearing loss. Patients with fibrous dysplasia develop trabecular bone overgrowth resulting in hearing loss if the lesions affect the temporal bones. Unfortunately, the mechanisms responsible for this hearing loss, which could be sensorineural and/or conductive, remain unclear. In this study, we used a unique transgenic mouse model of increased Gs G-protein coupled receptor (GPCR) signaling induced by expression of an engineered receptor, Rs1, in osteoblastic cells. These ColI(2.3)+/Rs1+ mice showed dramatic bone lesions that histologically and radiologically resembled fibrous dysplasia. We found that ColI(2.3)+/Rs1+ mice showed progressive and severe conductive hearing loss. Ossicular chain impingement increased with the size and number of dysplastic lesions. While sensorineural structures were unaffected, ColI(2.3)+/Rs1+ cochleae had abnormally high osteoclast activity, together with elevated tartrate resistant acid phosphatase (TRAP) activity and receptor activator of nuclear factor kappa-B ligand (Rankl) mRNA expression. ColI(2.3)+/Rs1+ cochleae also showed decreased expression of Sclerostin (Sost), an antagonist of the Wnt signaling pathway that normally increases bone formation. The osteocyte canalicular networks of ColI(2.3)+/Rs1+ cochleae were disrupted and showed abnormal osteocyte morphology. The osteocytes in the ColI(2.3)+/Rs1+ cochleae showed increased expression of matrix metalloproteinase 13 (MMP-13) and TRAP, both of which can support osteocyte-mediated peri-lacunar remodeling. Thus, while the ossicular chain impingement is sufficient to account for the progressive hearing loss in fibrous dysplasia, the deregulation of bone remodeling extends to the cochlea as well. Our findings suggest that factors regulating bone remodeling, including peri-lacunar remodeling by osteocytes, may be useful targets for treating the bony overgrowths and hearing changes of fibrous dysplasia and other bony pathologies.
CCN2, also known as connective tissue growth factor, is a member of the CCN (CCN1–6) family of modular matricellular proteins. Analysis of CCN2 function in vivo has focused primarily on its key role ...as a mediator of excess ECM synthesis in multiple fibrotic diseases. However, CCN2 and related family members are widely expressed during development. Recent studies using new genetic models are revealing that CCN2 has essential roles in the development of many tissues. This review focuses on current and emerging data on CCN2 and its functions in chondrogenesis and angiogenesis, and on new studies showing that CCN2 has essential functions during embryonic and postnatal development in a number of epithelial tissues.
Through a process called perilacunar remodeling, bone-embedded osteocytes dynamically resorb and replace the surrounding perilacunar bone matrix to maintain mineral homeostasis. The vital canalicular ...networks required for osteocyte nourishment and communication, as well as the exquisitely organized bone extracellular matrix, also depend upon perilacunar remodeling. Nonetheless, many questions remain about the regulation of perilacunar remodeling and its role in skeletal disease. Here, we find that suppression of osteocyte-driven perilacunar remodeling, a fundamental cellular mechanism, plays a critical role in the glucocorticoid-induced osteonecrosis. In glucocorticoid-treated mice, we find that glucocorticoids coordinately suppress expression of several proteases required for perilacunar remodeling while causing degeneration of the osteocyte lacunocanalicular network, collagen disorganization, and matrix hypermineralization; all of which are apparent in human osteonecrotic lesions. Thus, osteocyte-mediated perilacunar remodeling maintains bone homeostasis, is dysregulated in skeletal disease, and may represent an attractive therapeutic target for the treatment of osteonecrosis.
Abstract Bone remodeling, a combination of bone resorption and formation, requires precise regulation of cellular and molecular signaling to maintain proper bone quality. Whereas osteoblasts deposit ...and osteoclasts resorb bone matrix, osteocytes both dynamically resorb and replace perilacunar bone matrix. Osteocytes secrete proteases like matrix metalloproteinase-13 (MMP13) to maintain the material quality of bone matrix through perilacunar remodeling (PLR). Deregulated bone remodeling impairs bone quality and can compromise hearing since the auditory transduction mechanism is within bone. Understanding the mechanisms regulating cochlear bone provide unique ways to assess bone quality independent of other aspects that contribute to bone mechanical behavior. Cochlear bone is singular in its regulation of remodeling by expressing high levels of osteoprotegerin. Since cochlear bone expresses a key PLR enzyme, MMP13, we examined whether cochlear bone relies on, or is protected from, osteocyte-mediated PLR to maintain hearing and bone quality using a mouse model lacking MMP13 (MMP13−/− ). We investigated the canalicular network, collagen organization, lacunar volume via micro-computed tomography, and dynamic histomorphometry. Despite finding defects in these hallmarks of PLR in MMP13−/− long bones, cochlear bone revealed no differences in these markers, nor hearing loss as measured by auditory brainstem response (ABR) or distortion product oto-acoustic emissions (DPOAE), between wild type and MMP13−/− mice. Dynamic histomorphometry revealed abundant PLR by tibial osteocytes, but near absence in cochlear bone. Cochlear suppression of PLR corresponds to repression of several key PLR genes in the cochlea relative to long bones. These data suggest cochlear bone uniquely maintains bone quality and hearing independent of MMP13-mediated osteocytic PLR. Furthermore, the cochlea employs parallel mechanisms to inhibit remodeling by osteoclasts and osteoblasts, and by osteocytes, to protect hearing. Understanding the cellular and molecular mechanisms that confer site-specific control of bone remodeling have the potential to elucidate new pathways that are deregulated in skeletal disease.
Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) primarily characterized by an elevated red blood cell count, consequently to clonal expansion of myeloid progenitor cells driven almost ...exclusively by a mutation of a specific single nucleotide in JAK2 exon 14 (JAK2-V617F). JAK2-V617F mutational burden therefore represents a surrogate measure for the size of the "liquid tumor" underlying PV, i.e. the amount of malignant cells in the peripheral blood. We previously reported on sustained molecular responses (MR) in PV patients on interferon alpha (IFNa) based therapies. Monitoring MR is crucial for understanding the therapeutic potential of IFNa and can be an important parameter in future PV patient care. Thus, highly sensitive laboratory techniques providing most accurate quantification at lowest limit of detection (LOD) are required. Here we evaluate four different methods for JAK2-V617F mutational burden monitoring, which are digital droplet PCR (ddPCR), next-generation sequencing (NGS), quantitative PCR (qPCR) and allele-specific PCR (AS-PCR). Moreover, using an NGS-based approach we investigate the patients' clonal composition by targeted re-sequencing involving 54 genes in longitudinal patient samples.
To determine the characteristics of the different methods for burden quantification, we benchmarked a ddPCR assay (Assay ID dHsaCP2000061/dHsaCP2000062, Bio-Rad, Hercules, CA) using serial dilutions of JAK2-V617F positive in JAK2-V617F negative gDNA. The assay showed an LOD of 0.01% JAK2-V617F with a false positivity rate of 0 mutation-positive droplets in 8 independent measurements of healthy donor gDNA. Next we quantified the JAK2-V617F burden in baseline and follow-up (FU) gDNAs from a PV patient cohort on IFNa-based therapy (n=51) using ddPCR, a qPCR-based assay (ipsogen MutaQuant, Qiagen, Hilden, Germany), an NGS-based approach (TruSight Myeloid Sequencing Panel, Illumina, San Diego, CA) and an AS-PCR assay described previously (Kralovics et al, Blood, 2006). Covering the full spectrum of allelic burden values, correlations with ddPCR-derived values were best for NGS (R2=0.998) followed by qPCR (R2=0.976) and AS-PCR (R2=0.951). While mutational burden values >10% JAK2-V617F showed high concordance between methods, JAK2-V617F burdens <10% exhibited significant deviations from the benchmarked ddPCR assay, suggesting that ddPCR is most suitable for monitoring residual disease in PV. Therefore, applying ddPCR, we next re-evaluated two PV patients with baseline JAK2-V617F burdens of 46% and 28%, respectively, both previously classified as complete MR based on JAK2-V617F undetectable by AS-PCR. For those two patients, ddPCR revealed an actual residual disease of 3.02% and 2.39% mutant allele burden at 138 and 68 weeks post initiation of IFNa therapy, respectively. Further monitoring of these patients showed a sustained decrease in mutant allele burden to 1.53% and 0.20% at weeks 217 and 171, respectively.
In addition to JAK2-V617F burden quantification, our NGS approach allowed for quantitative evaluation of mutations in 54 genes implicated in myeloid diseases. After somatic variant calling, paired analysis of baseline and FU samples (n=96) from 48 PV patients revealed an average of 2.8 variants per patient in a total of 17 genes. Besides JAK2, recurrently affected genes were TET2, PHF6, ASXL1 and CEBPA. The paired analysis allowed for tracking changes in clonal structures upon therapy. Most notably, TET2-JAK2 double-positive clones present in two patients showed mutant burden decreases of 13% and 59% respectively, arguing against an implication of TET2 mutations in IFNa resistance. Furthermore, PHF6 positive clones present in three patients showed ambiguity upon therapy, shrinking concordantly with the JAK2 positive clone in two patients while expanding as a JAK2-independent clone in the third patient.
In summary, while larger studies will be required to assign statistical significance to mutations implicated in IFNa-based therapies, targeted re-sequencing of serial samples in MPN patient cohorts has the potential to deconvolute the impact of the patients' clonal architecture on therapeutic success. Moreover, as medications such as IFNa-based drugs have been shown to induce deep MR in a substantial fraction of PV patients, the use of highly accurate assays such as ddPCR will be crucial for monitoring minimal residual disease.
Klade:AOP Orphan: Employment. Gisslinger:Baxalta: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; AOP Orphan: Consultancy, Honoraria. Hoermann:Novartis: Honoraria; Ariad: Honoraria; Gilead: Research Funding; Amgen: Honoraria. Kralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees.
β1 integrin has been shown to contribute to vascular smooth muscle cell differentiation, adhesion and mechanosensation in vitro. Here we showed that deletion of β1 integrin at the onset of smooth ...muscle differentiation resulted in interrupted aortic arch, aneurysms and failure to assemble extracellular matrix proteins. These defects result in lethality prior to birth. Our data indicates that β1 integrin is not required for the acquisition, but it is essential for the maintenance of the smooth muscle cell phenotype, as levels of critical smooth muscle proteins are gradually reduced in mutant mice. Furthermore, while deposition of extracellular matrix was not affected, its structure was disrupted. Interestingly, defects in extracellular matrix and vascular wall assembly, were restricted to the aortic arch and its branches, compromising the brachiocephalic and carotid arteries and to the exclusion of the descending aorta. Additional analysis of β1 integrin in the pharyngeal arch smooth muscle progenitors was performed using wnt1Cre. Neural crest cells deleted for β1 integrin were able to migrate to the pharyngeal arches and associate with endothelial lined arteries; but exhibited vascular remodeling defects and early lethality. This work demonstrates that β1 integrin is dispensable for migration and initiation of the smooth muscle differentiation program, however, it is essential for remodeling of the pharyngeal arch arteries and for the assembly of the vessel wall of their derivatives. It further establishes a critical role of β1 integrin in the protection against aneurysms that is particularly confined to the ascending aorta and its branches.
► β1 integrin is crucial to the assembly of vascular extracellular matrix. ► β1 integrin is required for the maintenance of differentiation. ► β1 integrin is not required for cranial neural crest migration.
Members of the C(Cyr61) C(CTGF) N(Nov) family of matricellular proteins are involved in multiple aspects of embryonic and postnatal development and disease pathologies. CCN2, or connective tissue ...growth factor, is the second member of the family and has gained importance due to its prominent role in fibrotic disease. CCN2 has been extensively studied both in vitro and in vivo, but its specific roles remain unclear due to discrepancies in cell specific signaling mechanisms and experimental design. The current use of CCN2 blocking antibodies in clinical trials to treat symptoms associated with fibrosis and kidney function during diabetes warrants a clearer understanding of the function of CCN2 during normal physiology in order anticipate side effects. In order to investigate the functions of CCN2 during development, we have further analyzed the Ccn2 global knockout mouse and found that CCN2 plays essential roles in angiogenesis during vascular remodeling and cellular stress during endochondral ossification. CCN2 plays a dual role in vascular remodeling, first through mediating platelet derived growth factor (PDGF) signaling between endothelial cells and pericytes, and second through inducing the secretion of provisional and permanent vascular basement membrane components. Further analysis of the ECM defect observed in the growth plates of Ccn2 mutant mice revealed that the loss of CCN2 results in increased endoplasmic reticulum (ER) stress. Ccn2 mutants also exhibited decreased Nuclear Factor κB (NFκB) and autophagy-mediated cellular survival. Conversely, the overexpression of CCN2 results in attenuated ER stress and increased cellular survival during chemically induced ER stress. These results highlight a novel protective role for CCN2 during chondrocyte differentiation. Taken together, these results demonstrate that CCN2 plays important physiological roles in vivo and these roles should be considered during therapeutic interventions.
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