Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Dec 26, 2015; 7(11): 1262-1273
Published online Dec 26, 2015. doi: 10.4252/wjsc.v7.i11.1262
Induction of CXC chemokines in human mesenchymal stem cells by stimulation with secreted frizzled-related proteins through non-canonical Wnt signaling
David S Bischoff, Jian-Hua Zhu, Nalini S Makhijani, Dean T Yamaguchi
David S Bischoff, Jian-Hua Zhu, Nalini S Makhijani, Dean T Yamaguchi, Research Service, VA Greater Los Angeles Healthcare System, Los Angeles, CA 91343, United States
David S Bischoff, Dean T Yamaguchi, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90073, United States
Author contributions: Bischoff DS performed many of the experiments, designed some of the experiments, analyzed the data and contributed to writing the manuscript; Zhu JH performed the majority of the experiments; Makhijani NS maintained cell lines; Yamaguchi DT coordinated the research, designed some of the experiments, interpreted the data and contributed to writing the manuscript.
Supported by Merit Review Award from the United States, Department of Veterans Affairs Biomedical Laboratory Research and Development Service of the VA Office of Research and Development, No. I01BX000170.
Institutional review board statement: Not applicable.
Institutional animal care and use committee statement: Not applicable.
Conflict-of-interest statement: To the best of our knowledge, no conflict of interest exists. All authors are employees of the United States Federal Government and the work is part of their official duties.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Dean T Yamaguchi, MD, PhD, Associate Chief of Staff, Research Service, VA Greater Los Angeles Healthcare System, 11301 Wilshire Blvd, Building, Los Angeles, CA 91343, United States. dean.yamaguchi@va.gov
Telephone: +1-310-2683459 Fax: +1-310-2684856
Received: May 28, 2015
Peer-review started: June 1, 2015
First decision: September 17, 2015
Revised: October 23, 2015
Accepted: November 23, 2015
Article in press: November 25, 2015
Published online: December 26, 2015
Processing time: 210 Days and 22 Hours
Abstract

AIM: To investigate the effect of secreted frizzled-related proteins (sFRPs) on CXC chemokine expression in human mesenchymal stem cells (hMSCs).

METHODS: CXC chemokines such as CXCL5 and CXCL8 are induced in hMSCs during differentiation with osteogenic differentiation medium (OGM) and may be involved in angiogenic stimulation during bone repair. hMSCs were treated with conditioned medium (CM) from L-cells expressing non-canonical Wnt5a protein, or with control CM from wild type L-cells, or directly with sFRPs for up to 10 d in culture. mRNA expression levels of both CXCL5 and CXCL8 were quantitated by real-time reverse transcriptase-polymerase chain reaction and secreted protein levels of these proteins determined by ELISA. Dose- (0-500 ng/mL) and time-response curves were generated for treatment with sFRP1. Signal transduction pathways were explored by western blot analysis with pan- or phosphorylation-specific antibodies, through use of specific pathway inhibitors, and through use of siRNAs targeting specific frizzled receptors (Fzd)-2 and 5 or the receptor tyrosine kinase-like orphan receptor-2 (RoR2) prior to treatment with sFRPs.

RESULTS: CM from L-cells expressing Wnt5a, a non-canonical Wnt, stimulated an increase in CXCL5 mRNA expression and protein secretion in comparison to control L-cell CM. sFRP1, which should inhibit both canonical and non-canonical Wnt signaling, surprisingly enhanced the expression of CXCL5 at 7 and 10 d. Dickkopf1, an inhibitor of canonical Wnt signaling prevented the sFRP-stimulated induction of CXCL5 and actually inhibited basal levels of CXCL5 expression at 7 but not at 10 d post treatment. In addition, all four sFRPs isoforms induced CXCL8 expression in a dose- and time-dependent manner with maximum expression at 7 d with treatment at 150 ng/mL. The largest increases in CXCL5 expression were seen from stimulation with sFRP1 or sFRP2. Analysis of mitogen-activated protein kinase signaling pathways in the presence of OGM showed sFRP1-induced phosphorylation of extracellular signal-regulated kinase (ERK) (p44/42) maximally at 5 min after sFRP1 addition, earlier than that found in OGM alone. Addition of a phospholipase C (PLC) inhibitor also prevented sFRP-stimulated increases in CXCL8 mRNA. siRNA technology targeting the Fzd-2 and 5 and the non-canonical Fzd co-receptor RoR2 also significantly decreased sFRP1/2-stimulated CXCL8 mRNA levels.

CONCLUSION: CXC chemokine expression in hMSCs is controlled in part by sFRPs signaling through non-canonical Wnt involving Fzd2/5 and the ERK and PLC pathways.

Keywords: CXC chemokines; Mesenchymal stem cell; Osteogenesis; Differentiation; Wnt signaling pathway; Frizzled-related protein; Frizzled receptors

Core tip: Chemokines have multiple functions during bone formation and fracture repair. The ELR+ chemokines classically have a role in blood vessel formation and were found to be stimulated by the non-canonical Wnt5a protein and also by soluble frizzled-related proteins (sFRPs) that are known inhibitors of both canonical and non-canonical Wnt signaling. This stimulation was mediated via the p44/42 extracellular signal-regulated kinase and phospholipase C pathways signaling through the non-canonical frizzled receptors 2 and 5. This is a newly identified role for the sFRPs in stimulation of ELR+ chemokines which may be involved in blood vessel formation during wound repair.