The function of ITGBL1 in the process of cartilage formation and its potential as a therapeutic agent for. We believe that ITGBL1 possesses dual functions that promote cartilage formation while simultaneously inhibiting catabolic signals in chondrocytes.
Introduction
This neural crest cell is a vertebrate-specific cell population that originates from the dorsal part of the neural tube. In addition, in the cranial region, the neural crest cell contributes to the formation of cartilage and bone of the cranial, facial, and pharyngeal skeleton.
1-2. Chondrogenesis
Outside-in activation, when the RGD peptide of the artificial ligand binds to the extracellular domain of the integrin, the closed dimer opens in the unfolded state (extended state). This ensures cell stabilization and allows not only immobilization on the ECM, but also signal transmission between the extracellular and intracellular regions.
1-3. ITGBL1(Integrin beta like protein 1)
Chapter 2. ITGBL1 modulates integrin activity to promote cartilage formation and protect against arthritis
We show that ITGBL1 functions as an inhibitor of integrin-ECM interaction that is critical for cartilage formation and OA development. Using a mouse model of surgically induced OA, we show that ITGBL1 can protect against the development of OA in articular cartilage.
2-2. Material and methods
The cells were spotted as 15 μl droplets on culture dishes and maintained for 6 days to induce chondrogenesis. After harvesting, the cells were resuspended in medium containing 5 mM DTT and incubated for 30 min. The cells were then centrifuged for 5 min at 70 x g, floating cells were removed, and the remaining cells were cultured on a coverslip coated with fibronectin.
Cells were treated with the integrin inhibitor BIO1211, obtustatin, or ATN-161 (TOCRIS) and incubated at 36°C for 12 h, followed by harvesting for RNA extraction. Mesenchymal cells were maintained as micromass culture for 10 h and infected with Ad-Itgbl1 for 1 h. Mouse experiments were approved by the Animal Care and Use Committee of Ajou University College of.
However, one mouse was lost due to unforeseen circumstances during the experiment, and thus the total number of mice was nine (n=9). The experiment was independently repeated a total of six times with two mice per experimental condition in each independent test, and thus the total number of mice was twelve (n=12). Osteophyte development was identified by Safranin-O staining, and osteophyte maturity was determined by measuring the thickness of the subchondral bone plate [76].
2-3. Results
Schematic illustrating ITGBL1 unilateral injection [contralateral region injected with mGFP (membrane-green fluorescent protein)] into two-cell-stage Xenopus embryos to examine the ITGBL1
High-magnification images of the ethmoidal plate (Eth in p´´´) in control and Itgbl1-MO-injected embryos. The chondrogenic process in facial cartilage begins with the condensation of migrating cranial neural crest cells. Then, prechondrocytes secrete cartilage-specific ECM molecules while changing their morphology to oval mature chondrocytes.
Control chondrocytes secreted cartilage-specific ECM and were well distributed with a typical oval shape (Fig. 2-1-5Q). However, ITGBL1-depleted cells failed to secrete ECM and maintained close contact with neighboring cells (Fig. 2-1-5R), indicating that. Fibronectin deposition in facial tissue was visualized by immunostaining after unilateral injection of Itgbl1-MO.
We also examined ITGBL1 localization in prechondrocytes by expressing FLAG-tagged ITGBL1 in Xenopus embryos. Furthermore, ectopic expression of ITGBL1 in embryonic prechondrocytes promoted chondrogenic ECM deposition earlier than in control prechondrocytes (Fig. 2-1-6U). ITGBL1 function in chondrogenic differentiation of human bone marrow stem cells (BMSCs) and mouse limb bud mesenchyme.
ITGBL1 and aggrecan (Acan) expression quantified by q-PCR analysis during chondrogenesis. B
After exploiting the in vivo capabilities of the Xenopus embryo to identify a novel regulator of cartilage development, we next sought to investigate its mechanism of action in chondrogenic differentiation of hBMSCs (bone marrow-derived mesenchymal stem cells). PCR experiments revealed that Itgbl1 expression in chondrogenic hBMSCs gradually increased and peaked at day 12 of differentiation (Fig. 2. When we performed siRNA-mediated Itgbl1 knockdown and induced chondrogenesis in hBMSCs , Itgbl1 expression was effectively reduced, although it was gradually recovered by day 5 (fig. S4A–B).
Alcian blue and COL2A1 immunostaining showed that control hBMSC pellets deposited chondrogenic ECM normally, whereas Itgbl1 knockdown reduced chondrogenic ECM and COL2A1 expression (Fig. 2-2-1C-D).
Chondrogenic gene expression in hBMSCs after ITGBL1 overexpression
We tested ITGBL1 function in promoting chondrogenesis in the ATDC5 cell line and mouse chondrogenic mesenchymes isolated from limb buds, as these cells are already specified to differentiate into chondrocytes. Furthermore, expression of Itgbl1 in chondrogenic ATDC5 cells prevented the expression of Col10a1, suggesting that hypertrophic differentiation is suppressed by ITGBL1 (Fig. These results suggest that ITGBL1 enhances chondrogenesis and that the function of ITGBL1 is well conserved in vertebrates, including humans, although this may not be a master regulator of chondrogenic differentiation.
Chondrogenic gene expression upon ITGBL1 overexpression with increasing doses in the absence of differentiation medium analyzed by RT-PCR.
As expected, ITGBL1 depletion increased cell size, whereas Itgbl1 overexpression caused cells to detach from collagen-coated plates (Figs. 2-3-2E, F). Likewise, ITGBL1 depletion increased focal adhesion in PC3 cells as shown by integrin-β1 antibody staining (Figs. 2-3-2G,H). Using an active-form-specific integrin-β1 antibody and fluorescence-activated cell sorting (FACS), we saw that Itgbl1 overexpression reduced the amount of active integrin-β1, whereas siRNA-mediated ITGBL1 depletion increased the amount of active integrin-β1 (Figs. 2-3-3I, J).
In co-immunoprecipitation assays, ITGBL1 binds strongly to integrin-β1 in the presence but not in the absence of Ca2+ (Figs. 2-3-3K). As expected, activation of integrins by Mn2+ ions almost completely reversed integrin-ECM interactions in cells overexpressing Itgbl1 (Fig. 2-4-2C, D). Furthermore, this reduction in integrin-ECM interaction was fully recovered by integrin activation by Mn2+ treatment (Fig. 2-4-4H, I).
We also confirmed that ITGBL1 functions similarly in hBMSCs and in prechondrocytes of Xenopus facial cartilage (Figs. 2-4-5J-M). However, simultaneous depletion of each integrin α subunit, which forms a heterodimer with integrin β1, and β1 subunit synergistically induced Sox9 expression to a similar extent (Figs. 2-5-3F). As expected, ectopic expression of Itgbl1 increased Sox9 expression synergistically with the depletion of each integrin subunit that we examined (Figs. 2–5–3G).
Integrins α1 and α5 are highly expressed along with integrin-β1 in human primary chondrocytes. I
Thus, we investigated the potential protective function of ITGBL1 in arthritis development using human chondrocytes. In contrast, overexpression of Itgbl1 reduced Mmp3 and Mmp13 expression, and these reductions were reversed by activation of integrins (Fig. 2-6-1B-D). The protective function of ITGBL1 against catabolic gene expression is likely due to integrin inactivation and subsequent reduction in fragmented ECM molecules binding to the chondrocytes.
Depletion of ITGBL1 significantly (p<0.05) accelerated the binding of 29-kDa Fn-fs compared to control cells (Figure 2. We next tested whether depletion of ITGBL1 causes cartilage damage in vitro and in a mouse model in vivo. We speculated that depletion of ITGBL1 in mouse knee joint cartilage can result in OA-like phenotypes.
Furthermore, overexpression of Itgbl1 restored the expression of Col2a1 and Sox9 and reduced the expression of Mmp3 and Mmp13 in IL-1β-treated mouse chondrocytes (Fig. We therefore collected articular cartilage tissue from human OA patients to analyze the expression of Itgbl1. We then directly examined the role of ITGBL1 in OA pathogenesis using the surgical destabilization of the medial meniscus (DMM) mouse model of osteoarthritis.
2-4. Discussion
Summary and Conclusions
As started with chapter 1, this part describes the basic knowledge necessary for the understanding of experiments used in this doctoral thesis. The experimental animals and signaling pathway for the study of the craniofacial development were briefly described. In addition, chondrogenesis and integrin involved in cartilage formation important for facial formation have also been described.
In chapter 2 I described the function and role of ITGBL1 in the RNAseq of the pharyngeal arch of Xenopus laevis. Based on several papers that early integrin-ECM interaction may be required to promote prechondrocyte condensation. And the interaction between integrin and ECM must be inhibited for further decondensation, ECM secretion and cell shape change to complete chondrogenesis.
In this process, ITGBL1 inhibits integrin-ECM interactions, and this integrin inhibition enables chondrogenesis after prechondrocyte condensation. Fragmented ECM in OA patients binds to integrin from chondrocytes, synovial fibroblasts and immune cells. The unique function of ITGBL1 as a secreted integrin inhibitor points towards new approaches for the treatment of integrin-mediated human diseases and destructive cartilage diseases.
Chondrogenic differentiation of micromass-cultured ATDC5 cells was analyzed by Alcian blue staining, and glycosaminoglycan (GAG) production was measured
Chondrogenic differentiation of micromass-cultured ATDC5 cells was analyzed by Alcian blue staining, and glycosaminoglycan (GAG) production was measured. Hypertrophic maturation markers were analyzed during chondrogenic differentiation of ATDC5 cells in micromass culture after ITGBL1 overexpression (G).
Acknowledgements
늦은 나이에 일을 시작할 때 많은 걱정과 아낌없는 지원을 해주신 부모님께 진심으로 감사드리며, 항상 걱정해주시고 묵묵히 지켜봐 주시고 든든한 동반자 역할을 해 준 남편에게도 감사하다는 말씀 전하고 싶습니다. 나도 똑같이하는 동안 나에게 의존했습니다. 나 그거하고 싶어. 모든 분들의 이름을 밝힐 수는 없지만 이 기회를 빌어 저를 도와주신 모든 분들께 감사의 말씀을 전하고 싶습니다. 마지막으로, 본 논문을 완성할 수 있도록 도와주신 교수님께 다시 한 번 감사의 말씀을 전하고 싶습니다.
Grenier, J., et al., The relationship between neural crest cells and cranial mesoderm during head muscle development. Lapadula, G., et al., Integrin expression on chondrocytes: correlations with level of cartilage damage in human osteoarthritis. Terpstra, L., et al., Reduced chondrocyte proliferation and chondrodysplasia in mice lacking integrin-related kinase in chondrocytes.
Segat, D., et al., Integrins alpha(6A)beta 1 and alpha(6B)beta 1 promote different stages of chondrogenic cell differentiation. Homandberg, G.A., et al., Antisense oligonucleotides against the integrin receptor subunit alpha(5) reduce fibronectin fragment-mediated cartilage chondrolysis. Kim, J.H., et al., Regulation of the catabolic cascade in osteoarthritis by the zinc-ZIP8-MTF1 axis.
Zhen, G., et al., Inhibition of TGF-beta signaling in subchondral bone mesenchymal stem cells attenuates osteoarthritis. Bonnard, C., et al., Mutations in IRX5 impair craniofacial development and germ cell migration via SDF1. Takagi, J., et al., Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling.
