Geometry sensing through POR1 regulates Rac1 activity controlling early osteoblast differentiation in response to nanofiber diameter

Abstract

Bone grafting procedures in the United States rely heavily upon autografts and allografts, which are donor-dependent, cause donor site pain, and can transmit disease. Synthetic bone grafts can reduce these risks; however, synthetics lack the bone differentiating (osteoinductive) abilities of auto- and allografts. Achieving innate osteoinductive properties of synthetics through surface modifications is currently under investigation. This study focuses on nanofibers, with emphasis on how fiber diameter and the potential curvature sensor POR1 affect the activation of the signalingmoleculesRac1 and Arf1, and leading to expression of alkaline phosphatase (ALP), an osteoinductive marker. Diameters of 0.1, 0.3, and 1.0 μm were compared against a flat control. The highest level of Rac1 activation was achieved on the smallest fibers (0.1 μm), a trend that was long in POR1 knockdowns. This supports the hypothesis that on small nanofibers, POR1 favorably binds to highly curved cell membranes, which allows Rac1 to subsequently dissociate and activate. When the curvature is insufficient to bind POR1, POR1 binds to inactive Rac1 and competitively inhibits its activation. Arf1 activation followed an opposite trend, with the largest nanofibers exhibiting the highest activity. This trend reinforces the known interaction between Rac1 and Arf1 through the GIT/PIX complex, an Arf1 GAP and Rac1 GEF, respectively. Large, (1.0μm), nanofibers demonstrated the highest ALP activity, indicating that ALP expression is inversely dependent on Rac1 activation. Knockdown of POR1 resulted in increased ALP activity across the substrates but without regard to the curvature sensing trend seen previously. Thus, POR1 senses curvature and increases Rac1 activity, which negatively regulates bone differentiation.

Polymer fiber-based models of connective tissue repair and healing

“…Cells respond to their 3D environment through cytoskeletal actin stress fibers[17] and focal adhesions which activate downstream signaling pathways[18]. This outside-in signaling is known to affect critical cell functions including adhesion, migration, morphology, proliferation, gene expression, and differentiation[18-22]. Accordingly, interactions between cells and their local environment play a significant role in both tissue homeostasis and wound repair[23].…”

Identification of Gli1‐interacting proteins during simvastatin‐stimulated osteogenic differentiation of bone marrow mesenchymal stem cells

“…Importantly, Rac1 has also been implicated in insulin signaling . POR1 senses curvature and increases the Rac1 activity, which negatively regulates bone differentiation …”

“…49 POR1 senses curvature and increases the Rac1 activity, which negatively regulates bone differentiation. 50 RAC/PAK1 is a major regulator of YAP1, a crucial molecule in Hippo signaling that plays key roles in cell proliferation, differentiation, and apoptosis. 51 Silencing of PSMC2 in osteosarcoma cell lines significantly suppressed cell proliferation, enhanced apoptosis, and accelerated G2/M phase and/or S-phase arrest.…”

Multiscale Poly-(ϵ-caprolactone) Scaffold Mimicking Non-linearity in Tendon Tissue Mechanics

“…In consideration of the results, it is suggested that a 264 rpm scaffold could be advantageous for tendon regenerative medicine applications based on the fiber architecture and corresponding toe region boundaries. Future directions include harnessing fiber architecture and diameter to align cells in an organized manner via the concept of bioinstructive geometry 17,18 to replace or augment growth factors. The Chinese-fingertrap scaffold design provides compliance, an organized fiber matrix, and hollow tubular structure, suggesting the strategy as an encouraging approach for applications beyond tendon tissue engineering, including ligament engineering, vascular engineering, 34 and nerve regeneration.…”

“…The proposed design purpose includes developing the scaffold architecture to mimic the nonlinear stress-strain curve of natural tendon and utilizing the nanofiber geometry to instruct cells 16–18 towards tenogenesis. The results of this project have a broad impact and promising project potential with the opportunity to span across various regenerative medicine applications, including vascular engineering, nerve regeneration, and ligament and tendon tissue engineering, to improve the clinical outcome and patient quality of life.…”