Intranuclear Actin Regulates Osteogenesis

Abstract

Depolymerization of the actin cytoskeleton induces nuclear trafficking of regulatory proteins and global effects on gene transcription. We here show that in mesenchymal stem cells (MSCs), cytochalasin D treatment causes rapid cofilin-/importin-9-dependent transfer of G-actin into the nucleus. The continued presence of intranuclear actin, which forms rod-like structures that stain with phalloidin, is associated with induction of robust expression of the osteogenic genes osterix and osteocalcin in a Runx2-dependent manner, and leads to acquisition of osteogenic phenotype. Adipogenic differentiation also occurs, but to a lesser degree. Intranuclear actin leads to nuclear export of Yes-associated protein (YAP); maintenance of nuclear YAP inhibits Runx2 initiation of osteogenesis. Injection of cytochalasin into the tibial marrow space of live mice results in abundant bone formation within the space of 1 week. In sum, increased intranuclear actin forces MSC into osteogenic lineage through controlling Runx2 activity; this process may be useful for clinical objectives of forming bone.

Keywords: Bone; Cofilin; Cytoskeleton; Importin 9; Mesenchymal stem cells; Runx2; Yes-associated protein.

Figure 1

Actin depolymerization initiates and enhances osteogenesis. Mouse marrow-derived mesenchymal stem cell (mdMSC) or human marrow-derived MSC (hMSC) were treated with CytoD (0.1 μg/ml) for indicated times. Except for panel (B), cells were cultured in osteogenic medium. (A): Control and CytoD-treated mdMSC stained with phalloidin, day 3. Scale bars = 25 μm. (B): mdMSC cultured in MEM; Osx and Ocn reverse transcriptase polymerase chain reaction. (C): mdMSC response to continuous CytoD, day 3; notations a, b, c ≠ control and differ from each other with p < 0.05. (D): ALP assay (at d1, control without CytoD = 5.1 nmol nNP/μg total protein per minutes) and ALP stain, mdMSC. (E): Osteocalcin (OCN) protein at 5 d, mdMSC. 3 experiments assessed for densitometry of Ocn, shown in graphs to the right, confirm a significant increase; *, p< 0.05. (F): hMSC response to CytoD, 3 days; *, p < 0.01. Abbreviations: ALP, alkaline phosphatase; CTL, control; CytoD, cytochalasin D; OCN, osteocalcin.

Figure 2

Actin transport into the nucleus is required for enhanced osteogenic gene expression after CytoD. (A): Nuclear and cytoplasmic IB, mouse marrow-derived mesenchymal stem cell (mdMSC) ± CytoD, 3 days. Densitometry of nuclear actin, shown in graphs to the right, confirms a significant increase compared with control, n = 3, *, p< 0.05. (B): CellLight Actin-RFP transfected human marrow-derived MSC imaged 8 hours after ± CytoD. Scale bars = 25μM. (C, D, G). Reverse transcriptase polymerase chain reaction analysis after siRNA treatment for cofilin1 (C), importin 9 (D) and importin 9 + cofilin 1 (G) ±CytoD for 3 days. For Panels (C), (D), and Alkaline phosphatase a, b ≠ control and differ from each other, p < 0.01. (E): Nuclear and cytoplasmic IB. Densitometry for nuclear actin blots reflect n = 3, *, p < 0.05. (F): Phalloidin stain. Scale bars = 25 μM. Abbreviations: CTL, control; CytoD, cytochalasin D; LDH, lactate dehydrogenase; PARP, polyribose polymerase.

Figure 3

Osteogenesis resulting from increased nuclear actin requires Runx2 activity. (A): Mouse marrow-derived mesenchymal stem cell (mdMSC), ± CytoD for 3 days at 24 hours after Runx2 siRNA treatment; a, b ≠ control and differ from each other, p < 0.01. (B, C): Nuclear and cytoplasmic IB, 3-day treatment of cytochalasin D after importin 9 knock-down. Densitometry of nuclear Yes-associated protein (YAP) is shown in (B, C), n = 3, *, p < 0.05. (D): Reverse transcriptase polymerase chain reaction (RT-PCR) analysis ± leptomycin B (5 ng/ml) and ± CytoD for 3 days; a, b ≠ control and differ from each other with p < 0.01. (E): mdMSC transfected with YAP construct or empty vector. IB, left panel. RT-PCR ± CytoD for 3 days. a, b ≠ control and differ from each other, p <0.01. (F): mdMSC treated with CytoD for 0 (3−), 3d (3+), or on the first day only (1+/2−). IB for nuclear YAP. Densitometry of nuclear YAP bands is shown, n = 3, *, p< 0.05. (G): 2D and 3D images. mdMSC ± CytoD for 3 days, phalloidin stain. Scale bars = 25 μm. Abbreviations: CTL, control; CytoD, cytochalasin D; LDH, lactate dehydrogenase; PARP, polyribose polymerase; YAP, Yes-associated protein.

Figure 4

Bone formation is induced by intra-tibial injection of cytochalasin D. (A): 3D images of cross and vertical sections of right tibia reconstructed from μCT. (B, C): Trabecular and cortical quantitative measurement for the same tibia as (A). (D): H&E staining for the right tibia with/without CytoD treatment. Asterisks indicate significant difference, *, p< 0.05; **, p < 0.01. Abbreviations: CTL, control; CytoD, cytochalasin D.