Pivotal Role of Tenascin-W (-N) in Postnatal Incisor Growth and Periodontal Ligament Remodeling

The continuously growing mouse incisor provides a fascinating model for studying stem cell regulation and organ renewal. In the incisor, epithelial and mesenchymal stem cells assure lifelong tooth growth. The epithelial stem cells reside in a niche known as the cervical loop. Mesenchymal stem cells are located in the nearby apical neurovascular bundle and in the neural plexus. So far, little is known about extracellular cues that are controlling incisor stem cell renewal and guidance. The extracellular matrix protein tenascin-W, also known as tenascin-N (TNN), is expressed in the mesenchyme of the pulp and of the periodontal ligament of the incisor, and is closely associated with collagen 3 fibers. Here, we report for the first time the phenotype of tenascin-W/TNN deficient mice, which in a C57BL/6N background exhibit a reduced body weight and lifespan. We found major defects in the alveolar bone and periodontal ligament of the growing rodent incisors, whereas molars were not affected. The alveolar bone around the incisor was replaced by a dense scar-like connective tissue, enriched with newly formed nerve fibers likely leading to periodontal pain, less food intake and reduced body weight. Using soft food to reduce mechanical load on the incisor partially rescued the phenotype. In situ hybridization and Gli1 reporter mouse experiments revealed decreased hedgehog signaling in the incisor mesenchymal stem cell compartment, which coordinates the development of mesenchymal stem cell niche. These results indicate that TNN deficiency in mice affects periodontal remodeling and increases nerve fiber branching. Through periodontal pain the food intake is reduced and the incisor renewal and the neurovascular sonic hedgehog secretion rate are reduced. In conclusion, tenascin-W/TNN seems to have a primary function in rapid periodontal tissue remodeling and a secondary function in mechanosensation.Peer reviewe

Optimizing antibody isotype interactions in antitumor immunity by complement activation for improved therapy of cancer

Monoclonal antibody-based immunotherapy has been widely used as a strategy to treat cancer. Successful treatment of B-cell lymphoma with the monoclonal antibody (mAb) Rituximab (RTX) in combination with chemotherapy has increased the survival of patients and minimized the side effects of the treatment. However, many patients do not react to the treatment with RTX or gain resistance quickly. Thus, strategies to enhance the tumor cell killing and improve the response rates of mAb-based immunotherapy are a fundamental goal. In this study, I use four different B-cell lymphoma cell lines grown into 3D structures, called spheroids, as a model organism. Those spheroids, which are closer to the in vivo situation of B-cell lymphoma in patients compared to conventional in vitro 2D cell cultures, in combination with RTX, are tested for the activation of effector functions to eliminate tumor cells and compared to experiments conducted in the same cell lines in 2D cell cultures. Moreover, the therapeutic mAb RTX is only approved by the FDA in an IgG1 isotype form. Here, I test different isotype forms of RTX on their efficacy to kill cancer cells by the complement system and also by the activation of monocytes to engulf them in the process of phagocytosis. Interestingly, the IgG3 isotype form of RTX can induce both effector functions most efficiently while the IgG1 isotype form, used in clinical approaches, is only second most efficient in eradicating cancer cells. In addition, when grown into spheroids, the efficacy of both effector functions is reduced compared to 2D cell cultures. Furthermore, the efficacy of the complement system to kill the different B-cell lymphoma cell lines was directly correlated with the expression of the complement regulatory surface protein CD59. By blocking CD59, the efficacy of the complement system could be partially enhanced when cells were treated in 2D cell cultures but not when grown into 3D spheroids. In addition, the antibody-dependent phagocytosis (ADP) of cancer cells by monocytes might correlate with the expression of the RTX target surface protein CD20. Also, the previous incubation of B-cell lymphoma cells with a chemotherapy agent can enhance the efficacy of ADP by presumably providing an “eat me” signal to the effector cells.  In summary, this work shows that the outcome of a treatment with RTX in B-cell lymphoma patients could be improved by the detection of the specific features of the cancer cells, for example the expression of CD59 and CD20 and the structure of the tumor. Moreover, the different isotypes of RTX can activate effector functions in different intensities. The IgG3 isotype form might be able to overcome resistance or lack of reaction to the treatment in B-cell lymphoma patients but further experiments will be needed to investigate these possibilities.

Optimizing antibody isotype interactions in antitumor immunity by complement activation for improved therapy of cancer

Monoclonal antibody-based immunotherapy has been widely used as a strategy to treat cancer. Successful treatment of B-cell lymphoma with the monoclonal antibody (mAb) Rituximab (RTX) in combination with chemotherapy has increased the survival of patients and minimized the side effects of the treatment. However, many patients do not react to the treatment with RTX or gain resistance quickly. Thus, strategies to enhance the tumor cell killing and improve the response rates of mAb-based immunotherapy are a fundamental goal. In this study, I use four different B-cell lymphoma cell lines grown into 3D structures, called spheroids, as a model organism. Those spheroids, which are closer to the in vivo situation of B-cell lymphoma in patients compared to conventional in vitro 2D cell cultures, in combination with RTX, are tested for the activation of effector functions to eliminate tumor cells and compared to experiments conducted in the same cell lines in 2D cell cultures. Moreover, the therapeutic mAb RTX is only approved by the FDA in an IgG1 isotype form. Here, I test different isotype forms of RTX on their efficacy to kill cancer cells by the complement system and also by the activation of monocytes to engulf them in the process of phagocytosis. Interestingly, the IgG3 isotype form of RTX can induce both effector functions most efficiently while the IgG1 isotype form, used in clinical approaches, is only second most efficient in eradicating cancer cells. In addition, when grown into spheroids, the efficacy of both effector functions is reduced compared to 2D cell cultures. Furthermore, the efficacy of the complement system to kill the different B-cell lymphoma cell lines was directly correlated with the expression of the complement regulatory surface protein CD59. By blocking CD59, the efficacy of the complement system could be partially enhanced when cells were treated in 2D cell cultures but not when grown into 3D spheroids. In addition, the antibody-dependent phagocytosis (ADP) of cancer cells by monocytes might correlate with the expression of the RTX target surface protein CD20. Also, the previous incubation of B-cell lymphoma cells with a chemotherapy agent can enhance the efficacy of ADP by presumably providing an “eat me” signal to the effector cells.  In summary, this work shows that the outcome of a treatment with RTX in B-cell lymphoma patients could be improved by the detection of the specific features of the cancer cells, for example the expression of CD59 and CD20 and the structure of the tumor. Moreover, the different isotypes of RTX can activate effector functions in different intensities. The IgG3 isotype form might be able to overcome resistance or lack of reaction to the treatment in B-cell lymphoma patients but further experiments will be needed to investigate these possibilities.

Insulin-like growth factor 1 receptor (IGF1R) signaling regulates osterix expression and cartilage matrix mineralization during endochondral ossification

Insulin-like growth factor I receptor (IGF1R) signaling is important for bone formation via endochondral ossification. Igf1r deficient mice show proportional dwarfism and alterations in chondrocyte proliferation, hypertrophy and apoptosis within the growth plate. In addition, gene ablation in mouse demonstrated that IGF1R signaling is important for osteoblast mediated bone mineralization. However, the mineralization in the terminal hypertrophic zone of the growth plate is also an essential step in endochondral ossification preceding bone formation. Therefore, we analyzed the influence of IGF1R signaling on this process by using mice with a specific deletion of Igf1r in chondrocytes. Studies in embryonic metatarsal explant cultures showed that mineralization of the terminal hypertrophic zone was strongly reduced when IGF1R signaling was lacking. This decreased mineralization may in part result from the delay in hypertrophic differentiation in the Igf1r deficient metatarsals. However, mineralization was impaired even stronger than hypertrophy, suggesting a mineralization promoting effect of IGF signaling that is independent of hypertrophic differentiation. We found a markedly decreased osterix expression suggesting that osterix is a downstream target of IGF1R in chondrocytes. MMP13 expression was strongly reduced in metatarsals lacking the IGF1R while alkaline phosphatase expression and activity were less affected. We conclude that endogenous IGF1R signaling is important for growth plate matrix remodeling and calcification leading to bone formation and suggest that regulation of osterix expression and its downstream target MMP13 are part of the underlying mechanism. (c) 2015 Elsevier Inc. All rights reserved

Exploring complement-dependent cytotoxicity by rituximab isotypes in 2D and 3D-cultured B-cell lymphoma

Background The therapeutic IgG1 anti-CD20 antibody, rituximab (RTX), has greatly improved prognosis of many B-cell malignancies. Despite its success, resistance has been reported and detailed knowledge of RTX mechanisms are lacking. Complement-dependent cytotoxicity (CDC) is one important mode of action of RTX. The aim of this study was to systematically evaluate factors influencing complement-mediated tumor cell killing by RTX. Methods Different RTX isotypes, IgG1, IgG3, IgA1 and IgA2 were evaluated and administered on four human CD20(+) B-cell lymphoma cell lines, displaying diverse expression of CD20 and complement-regulatory protein CD59. Complement activation was assessed on lymphoma cells grown in 2 and 3-dimensional (3D) culture systems by trypan blue exclusion. CDC in 3D spheroids was additionally analyzed by Annexin V and propidium iodide staining by flow cytometry, and confocal imaging. Anti-CD59 antibody was used to evaluate influence of CD59 in RTX-mediated CDC responses. Statistical differences were determined by one-way ANOVA and Tukey post hoc test. Results We found that 3 out of 4 lymphomas were sensitive to RTX-mediated CDC when cultured in 2D, while 2 out of 4 when grown in 3D. RTX-IgG3 had the greatest CDC potential, followed by clinical standard RTX-IgG1 and RTX-IgA2, whereas RTX-IgA1 displayed no complement activation. Although the pattern of different RTX isotypes to induce CDC were similar in the sensitive lymphomas, the degree of cell killing differed. A greater CDC activity was seen in lymphoma cells with a higher CD20/CD59 expression ratio. These lymphomas were also sensitive to RTX when grown in 3D spheroids, although the CDC activity was substantially reduced compared to 2D cultures. Analysis of RTX-treated spheroids demonstrated apoptosis and necrosis essentially in the outer cell-layers. Neutralization of CD59 overcame resistance to RTX-mediated CDC in 2D-cultured lymphoma cells, but not in spheroids. Conclusions The results demonstrate that CDC outcome in CD20(+) B-cell lymphoma is synergistically influenced by choice of RTX isotype, antigen density, tumor structure, and degree of CD59 expression. Assessment of tumor signatures, such as CD20/CD59 ratio, can be advantageous to predict CDC efficiency of RTX in vivo and may help to develop rational mAbs to raise response rates in patients

Collagen II regulates chondroycte integrin expression profile and differentiation

Collagen II is the major fibril-forming collagen in cartilage. Complete absence of collagen II in mice is not compatible with life and in humans mutations in the COL2A1 gene lead to osteochondrodysplasias with diverse phenotypes. However, mechanistic studies on how chondrocytes respond to a lack of collagen II in their extracellular matrix are limited. Primary mouse chondrocytes were isolated from knee joints of newborn mice and transfected with siRNA targeting Col2 alpha(1) to suppress collagen II expression. The expression of integrin receptors and matrix proteins was investigated by RT-PCR and immunoblots. The localization of matrix components was evaluated by immunostaining. Signaling pathways and the differentiation state of chondrocytes was monitored by RT-PCR and flow cytometry. We demonstrate that in the absence of collagen II chondrocytes start to produce collagen I. Some binding partners of collagen II are partially lost from the matrix while other proteins, e.g. COMP, were still found associated with the newly formed collagen network. The lack of collagen II induced changes in the expression profile of integrins. Further, we detected alterations in the Indian hedgehog/parathyroid hormone-related protein (lhh/PTHrP) pathway that were accompanied by changes in the differentiation state of chondrocytes. Collagen II seems not to be essential for chondrocyte survival in culture but it plays an important role in maintaining chondrocyte differentiation. We suggest that a crosstalk between extracellular matrix and cells via integrins and the Ihh/PTHrP pathway is involved in regulating the differentiation state of chondrocytes

Impact of Arginine to Cysteine Mutations in Collagen II on Protein Secretion and Cell Survival

Inherited point mutations in collagen II in humans affecting mainly cartilage are broadly classified as chondrodysplasias. Most mutations occur in the glycine (Gly) of the Gly-X-Y repeats leading to destabilization of the triple helix. Arginine to cysteine substitutions that occur at either the X or Y position within the Gly-X-Y cause different phenotypes like Stickler syndrome and congenital spondyloepiphyseal dysplasia (SEDC). We investigated the consequences of arginine to cysteine substitutions (X or Y position within the Gly-X-Y) towards the N and C terminus of the triple helix. Protein expression and its secretion trafficking were analyzed. Substitutions R75C, R134C and R704C did not alter the thermal stability with respect to wild type; R740C and R789C proteins displayed significantly reduced melting temperatures (T-m) affecting thermal stability. Additionally, R740C and R789C were susceptible to proteases; in cell culture, R789C protein was further cleaved by matrix metalloproteinases (MMPs) resulting in expression of only a truncated fragment affecting its secretion and intracellular retention. Retention of misfolded R740C and R789C proteins triggered an ER stress response leading to apoptosis of the expressing cells. Arginine to cysteine mutations towards the C-terminus of the triple helix had a deleterious effect, whereas mutations towards the N-terminus of the triple helix (R75C and R134C) and R704C had less impact

COMP in the Infrapatellar Fat Pad-Results of a Prospective Histological, Immunohistological, and Biochemical Case-Control Study

Knee osteoarthritis (OA) involves several structures and molecules in the joint, which interact in a pathophysiological process. One of these molecules is the cartilage oligomeric matrix protein (COMP). Elevated COMP levels in the synovial fluid as well as in the serum have been described in OA patients. However, this has not been described in the infrapatellar fat pad (IPFP) tissue before. In this prospective trial, we collected 14 IPFPs from patients with high-grade OA (mean age 63.8 +/- 17.6 years) who underwent total knee replacement (OA group) and from 11 healthy patients (mean age 33.7 +/- 14.8 years) who underwent anterior cruciate ligament reconstruction (control group). The presence of macrophages (CD68 and CD206) and proinflammatory cytokines (interleukin 1 beta [IL-1 beta] and IL-6) was analyzed. Histological and immunohistological examinations as well as immunoblotting analysis for COMP, leptin, and matrix-metalloproteinase-3 were performed. The IPFPs of both the OA and control group consisted of adipose tissue and fibrous tissue, and the fibrous tissue showed higher score values than the adipose tissue for COMP staining (intensity as well as stained area) in both groups. Although COMP could be detected in most samples, leptin expression was found only in single specimens. COMP could be detected mostly in the fibrous tissue portion of the IPFP. We speculate that it is involved in a remodeling process taking place in the IPFP during OA. Presence of leptin was irregular in immunohistology, and the control group showed higher scores in case of presence. Interestingly, immunoblotting could detect leptin in all analyzed samples. (c) 2019 The Authors. Journal of Orthopaedic Research (R) published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Societ

Ablation of the miRNA cluster 24 in cartilage and osteoblasts impairs bone remodeling

MicroRNAs (miRNAs) post-transcriptionally regulate cartilage and bone development and function, however, only few miRNAs have been described to play a role for cartilage to bone transition in vivo. Previously, we showed that cartilage-specific deletion of the Mirc24 cluster in newborn male mice leads to impaired growth plate cartilage development due to increased RAF/MEK/ERK signaling and affects the stability of the cartilage extracellular matrix on account of decreased SOX6 and SOX9 and increased MMP13 levels. Here, we studied how Mirc24 cluster inactivation in cartilage and osteoblasts leads to an increased bone density associated with defects in collagen remodeling in trabecular bone. No changes in osteoblast distribution were observed, whereas the number of osteoclasts was reduced and TRAP activity in osteoclasts decreased. Surprisingly, an increased level of cluster-encoded miR-322 or miR-503 raises Rankl gene expression and inactivation of the cluster in chondrocytes reduces Rankl expression. These results suggest that the Mirc24 cluster regulates Rankl expression in chondrocytes at the chondro-osseous border, where the cluster is mainly expressed to modulate osteoclast formation, bone remodeling and bone integrity