Integrin engagement regulates monocyte differentiation through the forkhead transcription factor Foxp1
Can Shi, Xiaobin Zhang, Zhiping Chen, Karina Sulaiman, Mark W. Feinberg, Christie M. Ballantyne, Mukesh K. Jain, Daniel I. Simon
Can Shi, Xiaobin Zhang, Zhiping Chen, Karina Sulaiman, Mark W. Feinberg, Christie M. Ballantyne, Mukesh K. Jain, Daniel I. Simon
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Article Vascular biology

Integrin engagement regulates monocyte differentiation through the forkhead transcription factor Foxp1

Abstract

The precise signals responsible for differentiation of blood-borne monocytes into tissue macrophages are incompletely defined. “Outside-in” signaling by integrins has been implicated in modulation of gene expression that affects cellular differentiation. Herein, using differential display PCR, we have cloned an 85-kDa forkhead transcription factor (termed Mac-1–regulated forkhead [MFH] and found subsequently to be identical to Foxp1) that is downregulated in β2-integrin Mac-1–clustered compared with Mac-1–nonclustered monocytic THP-1 cells. MFH/Foxp1 is expressed in untreated HL60 cells, and its expression was markedly reduced during phorbol ester–induced monocyte differentiation, but not retinoic acid–induced granulocyte differentiation. Overexpression of MFH/Foxp1 markedly attenuated phorbol ester–induced expression of c-fms, which encodes the M-CSF receptor and is obligatory for macrophage differentiation. This was accompanied by decreased CD11b expression, cell adhesiveness, and phagocytosis. Using electromobility shift and reporter assays, we have established that MFH/Foxp1 binds to previously uncharacterized sites within the c-fms promoter and functions as a transcriptional repressor. Deficiency of Mac-1 is associated with altered regulation of MFH/Foxp1 and monocyte maturation in vivo. Taken together, these observations suggest that Mac-1 engagement orchestrates monocyte-differentiation signals by regulating the expression of the forkhead transcription repressor MFH/Foxp1. This represents a new pathway for integrin-dependent modulation of gene expression and control of cellular differentiation.

Authors

Can Shi, Xiaobin Zhang, Zhiping Chen, Karina Sulaiman, Mark W. Feinberg, Christie M. Ballantyne, Mukesh K. Jain, Daniel I. Simon

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Clustering of Mac-1 downregulates expression of 12CC4/MFH/Foxp1. (A) Clu...
Clustering of Mac-1 downregulates expression of 12CC4/MFH/Foxp1. (A) Clustering of Mac-1. Cytokine-treated THP-1 cells were added to fibrinogen-coated and gelatin-blocked wells. Adhesion was promoted by the addition of the anti-CD18 stimulating mAb KIM127 (5 μg/ml) in the presence and absence of the anti-CD11b mAb LPM19c (10 μg/ml). After washing, adhesion was quantified by measurement of the fluorescence of 2′,7′-bis-(2-carboyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester–loaded (BCECF AM-loaded) THP-1 cells (Molecular Probes, Eugene, Oregon, USA). Triplicate determination (mean ± SD) representative of three separate experiments is shown. (B) Clustering of Mac-1 downregulates expression of 12CC4. Mac-1–dependent gene expression was examined using DD-PCR with RNA from Mac-1–clustered (+) and –nonclustered (–) THP-1 cells. cDNA expression patterns were analyzed from three independent clustering experiments. The arrow designates a band (12CC4) with reduced intensity after clustering in all three experiments. (C) Northern analysis. Northern blots containing 1 μg/lane of mRNA isolated from Mac-1–clustered and –nonclustered THP-1 cells were probed with 32P-labeled 12CC4 cDNA. RNA size is indicated in kilobases. (D) In vitro transcription and translation of vector alone and 12CC4. In vitro–coupled transcription and translation of native full-length 12CC4 (4,954 bp) produced a single band of predicted size (molecular weight ∼85,000).