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Jie Chao, Xiaoniu Dai, Tiffany Peña, David A. Doyle, Timothy M

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1 MCPIP1 Regulates Fibroblast Migration in 3-D Collagen Matrices Downstream of MAP Kinases and NF-κB 
Jie Chao, Xiaoniu Dai, Tiffany Peña, David A. Doyle, Timothy M. Guenther, Mark A. Carlson  Journal of Investigative Dermatology  Volume 135, Issue 12, Pages (December 2015) DOI: /jid Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Effect of fibroblast-populated three-dimensional (3-D) collagen matrix (FPCM) release on fibroblast migration and expression of monocyte chemotactic protein–induced protein 1 (MCPIP1). (a) Migration of green fluorescent protein (GFP)-expressing fibroblasts out of nested matrices was decreased 24hours after matrix release. Fibroblast migration shown at the interface between the nested matrix and the restrained cell-free matrix. Left scale bar=200μm, right scale bar=80μm. (b) Plot of migration (three separate experiments from panel a). (c) MCPIP1 induction after FPCM release. Whole-cell lysates from attached or released matrices immunoblotted for MCPIP1 and β-actin. (d) MCPIP1 densitometry (four separate experiments from panel c). (e) MCPIP1 immunocytochemistry in the attached versus released FPCM. Blue=DAPI; green=MCPIP1. Scale bar=20μm. Data are mean±SEM.; *P<0.05 versus attached (unpaired t-test). Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Effect of monocyte chemotactic protein–induced protein 1 (MCPIP1) RNAi or ectopic expression on matrix contraction, matrix cell number, and fibroblast migration. (a) Immunoblots of lysates from fibroblast-populated three-dimensional (3-D) collagen matrix (FPCMs) expressing siRNA (MCPIP1 versus nonsense). (b and c) Effect of MCPIP1 RNAi on FPCM contraction (well diameter=19mm); plot=three experiments. (d) Effect of MCPIP1 RNAi on FPCM cell number, 1-day post release. (e and f) Effect of MCPIP1 RNAi on migration out of the released, nested FPCM (scale bar=80μm); plot=three experiments. (g) Immunoblots of lysates from FPCMs expressing MCPIP1-Flag versus Flag. (h and i) Effect of MCPIP1-Flag expression on FPCM contraction; plot=three experiments. (j) Effect of MCPIP1-Flag expression on FPCM cell number, 1-day post release. (k and l) Effect of MCPIP1-Flag expression on migration out of the attached, nested FPCM (scale bar=80μm); plot=three experiments. *P<0.05, unpaired t-test. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Kinase activity, inhibition, monocyte chemotactic protein–induced protein 1 (MCPIP1) expression, and migration in the fibroblast-populated three-dimensional (3-D) collagen matrix (FPCM). (a–f) p38, Erk1/2, c-Jun N-terminal kinase (JNK), and Akt phosphorylation after FPCM release (immunoblots of whole lysates), with densitometry of n=4 experiments. (g–j) Effect of kinase inhibitors (JNK=SP600125, MEK=U0126, p38=SB203580, PI3K=LY294002) on FPCM MCPIP1, 30minutes and 24hours post release (whole-lysate immunoblots), with densitometry (n=4 experiments each). *P<0.05 versus vehicle-attached (unpaired t-test); #P<0.05 versus vehicle-released (unpaired t-test). (k and l) Effect of kinase inhibitors on migration out of the released, nested matrix (upper scale bar=200μm, lower scale bar=80μm); plot represents n=3 experiments/condition. *P<0.05, unpaired t-test. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 NF-κB signaling, monocyte chemotactic protein–induced protein 1 (MCPIP1) induction, and inhibition of migration in the fibroblast-populated three-dimensional (3-D) collagen matrix (FPCM). (a and b) NF-κB p65 phosphorylation after FPCM release (nuclear fraction immunoblots); n=4 experiments for densitometry. (c and d) Effect of kinase inhibitors on p-p65 in attached versus 20-minute-released FPCMs; n=4 experiments for densitometry. (e) NF-κB p65 binding sequence, MCPIP1 promoter region. (f) Chromatin immunoprecipitation of p65 binding to the MCPIP1 promoter, 20minutes after FPCM release. (g and h) Effect of NF-κB activation inhibitor (SC-514) on MCPIP1 induction after FPCM release (24-hour time point; whole-cell immunoblots); n=4 experiments for densitometry. (i) Effect of SC-514 resulted on migration out of the nested released FPCM (immunofluorescent images in Supplementary Figure S1C and D online); n=3 experiments. *P<0.05, unpaired t-test. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Monocyte chemotactic protein–induced protein 1 (MCPIP1) and p53 association after fibroblast-populated three-dimensional (3-D) collagen matrix (FPCM) release. (a) MCPIP1 and p53 immunocytochemistry in attached versus 1day-released FPCM. Blue=DAPI; green=MCPIP1; red=p53; scale bar=20μm. (b and c) MCPIP1 and p53 co-localization after FPCM release (whole lysates from attached versus 1day-released FPCMs immunoprecipitated for MCPIP1 or p53 and then immunoblotted for the other; IgG used as immunoprecipitate control). (d–f) Effect of RNAi of MCPIP1 or p53 on p53 or MCPIP1 in 1day-released FPCM (whole-lysate immunoblots); n=4 experiments for densitometry. *P<0.05 versus attached (unpaired t-test). Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Monocyte chemotactic protein–induced protein 1 (MCPIP1) regulates cell migration through p53. (a–c) Effect of MCPIP1-Flag expression and/or p53 RNAi on MCPIP1 and p53 in attached versus 1day-released fibroblast-populated three-dimensional (3-D) collagen matrix (FPCM; whole-lysate immunoblots); n=4 experiments for densitometry. (d and e) Effect of MCPIP1-Flag expression and/or p53 RNAi on migration out of attached versus 1day-released FPCMs (scale bar=80μm); plot represents n=3 experiments/condition. *P<0.05 versus control-attached (unpaired t-test); #P<0.05 versus control-released (unpaired t-test). (f and g) The Putative pathway of the MCPIP1-centric pathway regulating migration in the FPCM. Stress release of the FPCM activates PI3K, ERK, p38, and c-Jun N-terminal kinase (JNK). Activated NK-κB induces expression of MCPIP1, which upregulates p53, which then inhibits fibroblast migration through unknown mechanisms. A pointed arrowhead head=stimulatory effect; flat arrowhead=inhibition. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

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