Activation of TGF-1/p38/Smad3 signaling in stromal cells requires ROS-mediated MMP-2 activity during bone marrow damage

¹ Department of Pediatrics, Robert C. Byrd Health Sciences Center, West Virginia University, School of Medicine, Morgantown, West Virginia
² Department of Microbiology, Immunology and Cell Biology, Robert C. Byrd Health Sciences Center, West Virginia University, School of Medicine, Morgantown, West Virginia
³ The Mary Babb Randolph Cancer Center, Robert C. Byrd Health Sciences Center, West Virginia University, School of Medicine, Morgantown, West Virginia
⁴ Department of Pediatrics, Department of Microbiology, Immunology and Cell Biology, and The Mary Babb Randolph Cancer Center, Robert C. Byrd Health Sciences Center, West Virginia University, School of Medicine, Morgantown, West Virginia

^* To whom correspondence should be addressed. E-mail: lgibson{at}hsc.wvu.edu.

	Abstract

Dose-escalated chemotherapy has proven utility in a variety of treatment settings, including preparative regimens prior to bone marrow or hematopoietic stem cell transplantation (BMT/HSCT). However, the potential damage imposed by aggressive regimens on the marrow microenvironment warrants further investigation. In the present study, we tested the hypothesis that dose-escalated chemotherapy, with etoposide as a model chemotherapeutic agent, activates the transforming growth factor beta-1 (TGF-1) signaling pathway in bone marrow stromal cells. Following high-dose etoposide exposure in vitro, Smad3 protein was phosphorylated in a time- and dose dependent manner in marrow derived stromal cells, coincident with the release of active and latent TGF-1 from the extracellular matrix (ECM). Phosphorylation was modulated by p38 kinase, with translocation of Smad3 from the cytoplasm to the nucleus subsequent to its phosphorylation. Etoposide-induced activation of TGF-1 followed the generation of reactive oxygen species (ROS) and required MMP-2 protein availability. Chemotherapy effects were diminished in MMP-2^-/- knockout stromal cells and TGF-1 knockdown siRNA-transfected stromal cells, in which phosphorylation of Smad3 was negligible following etoposide exposure. Stable transfection of a human MMP-2 cDNA into bone marrow stromal cells resulted in elevated phosphorylation of Smad3 during chemotherapy. These data suggest TGF-1/p38/Smad3 signaling cascades are activated in bone marrow stromal cells following dose-escalation chemotherapy, and may contribute to chemotherapy-induced alterations of the marrow microenvironment.

Key Words. TGF-1, Smad3, ROS, MMP-2, Chemotherapy, Microenvironment

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