Dataset Information

Substrate stiffness regulates the differentiation profile and functions of osteoclasts via cytoskeletal arrangement.

ABSTRACT:

Objectives

Aging and common diseases alter the stiffness of bone tissue, causing changes to the microenvironment of the mechanosensitive bone cells. Osteoclasts, the sole bone-resorbing cells, play a vital role in bone remodeling. This study was performed to elucidate the mechanism through which osteoclasts sense and react to substrate stiffness signals.

Materials and methods

We fabricated polydimethylsiloxane (PDMS) substrates of different stiffness degrees for osteoclast formation progressed from osteoclast precursors including bone marrow-derived macrophages (BMMs) and RAW264.7 monocytes. Osteoclast differentiation in response to the stiffness signals was determined by examining the cell morphology, fusion/fission activities, transcriptional profile, and resorption function. Cytoskeletal changes and mechanosensitive adhesion molecules were also assessed.

Results

Stiffer PDMS substrates accelerated osteoclast differentiation, firstly observed by variations in their morphology and fusion/fission activities. Upregulation of canonical osteoclast markers (Nfatc1, Acp5, Ctsk, Camk2a, Mmp9, Rela, and Traf6) and the fusion master regulator DC-stamp were detected on stiffer substrates, with similar increases in their bone resorption functions. Additionally, the activation of cytoskeleton-associated adhesion molecules, including fibronectin and integrin αvβ3, followed by biochemical signaling cascades of paxillin, FAK, PKC, and RhoA, was detected on the stiffer substrates.

Conclusions

This is the first study to provide evidence proving that extracellular substrate stiffness is a strong determinant of osteoclast differentiation and functions. Higher stiffness upregulated the differentiation profile and activity of osteoclasts, revealing the mechanical regulation of osteoclast activity in bone homeostasis and diseases.

SUBMITTER: Wang Q

PROVIDER: S-EPMC8780927 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Treating recurrent GBM is a clinical challenge due to its highly resistant and aggressive nature. In order to develop new therapeutic targets for recurrent GBM a better understanding of its molecular landscape is necessary. Here we used a cellular model, developed in our lab which generates paired primary and recurrent samples from GBM cell lines and primary patient samples hence allowing us to compare the molecular differences between the two populations. Total RNA seq analysis of parent and recurrent population of two cell lines and one patient sample revealed a significant upregulation of Extracellular matrix interaction in recurrent population. Since matrix stiffness plays a pivotal role in cell-ECM interaction and downstream signaling, we developed a system that mimicked the brain like substrate stiffness by using collagen coated polyacrylamide-based substrate whose stiffness can be modified from normal brain (0.5kPa) to tumorigenic (10kPa). Using these substrates, we were able to capture the morphological and physiological differences between parent and recurrent GBM which were not evident on plastic surfaces (~1 GPa). On 0.5kPa, unlike circular parent cells, recurrent GBM cells showed two morphologies (circular and elongated). The recurrent cells growing on 0.5kPa also showed higher proliferation, invasion, migration and in-vivo tumorigenicity in orthotropic GBM mouse model, compared to parent cells. Furthermore, recurrent cells exhibited elevated velocity irrespective of substrate stiffness, which indicated that recurrent cells may possess inherent differential mechanosignalling ability which was reflected by higher expression of ECM proteins like Collagen IVA, MMP2 and MMP9. Moreover, mice brain injected with recurrent cells grown on 0.5kPa substrate showed higher Young’s modulus values suggesting that recurrent cells conditioned on 0.5kPa make the surrounding ECM stiffer. Importantly, inhibition of EGFR signaling, that is amplified with tissue stiffening in GBM resulted in decreased invasion, migration and proliferation in 0.5kPa recurrent cells, but interestingly survival remained unaffected, highlighting the importance of mimicking the physiological stiffness of the brain mimicking clinical scenario. Total RNA seq analysis of parent and recurrent cells grown on plastic and 0.5kPa substrate identified PLEKHA7 as significantly upregulated gene specifically in 0.5kPa recurrent sample. Higher protein expression of PLEKHA7 in recurrent GBM as compared to primary GBM was validated in patient biopsies. Accordingly, PLEKHA7 knockdown reduced invasion and survival of recurrent GBM cells. Together, these data provides a model system that captures the differential mechanosensing signals of primary and recurrent GBM cells and identifies a novel potential target specific for recurrent GBM.