Signaling molecules that bind to chemokine receptors should play key roles in regulation of cell migration induced by chemokines. To characterize the CCR1-mediated cellular signal transduction mechanism, we used the yeast two-hybrid system to identify a cellular ligand for CCR1. LZIP, which has been known as a transcription factor in various cell types, was identified as a CCR1 binding protein. Although the ability of LZIP to bind DNA is possibly what allows it to function as a transcription factor, its detailed function and participation in chemotaxis have not been established. We found that LZIP binds to CCR1 based on results of a mammalian two-hybrid assay and immunoprecipitation experiments. The 21-260 residues of LZIP were essential for interaction with CCR1. Results from a chemotaxis assay using LZIP transfected cells showed that LZIP enhanced Lkn-1-induced chemotaxis, whereas the chemotactic activities induced by other CC chemokines that bind to CCR1, including MIP-1alpha, RANTES, or HCC-4, were not affected by LZIP overexpression. These data indicate that LZIP binds to CCR1 and that the interaction between CCR1 and LZIP participates in regulation of Lkn-1-dependent cell migration without affecting the chemotactic activities of other CC chemokines that bind to CCR1.
Human LZIP is a transcription factor that is involved in leukocyte cell mobility. Expression of LZIP is known to differentially regulate monocyte cell migration induced by CCR1-dependent chemokines. However, its transcriptional regulation has not been characterized. Our results indicate that Lkn-1 induces LZIP expression in a time-and dose-dependent manner, and the induction of LZIP shows an immediate early response to Lkn-1. We identified and cloned ϳ1.4 kb of the LZIP promoter from a human genomic DNA. To identify regulatory elements controlling restricted expression of LZIP, deletion mutants were constructed from the 1469-bp LZIP promoter region (؊1219/؉251) linked to the luciferase reporter gene. Maximal promoter activity was contained within 613 bp from the tentative transcription initiation site and was sharply reduced in a truncated construct (؊338/؉251). This promoter sequence contained consensus NF-B-and Sp-1-binding sites. Results from an inhibitor assay showed that NF-B is involved in Lkn-1-induced LZIP expression, but Sp-1 is not. We also demonstrated that NF-B binds to the LZIP promoter and that the binding is specific, as revealed by an electrophoretic mobility shift assay and a mutation analysis. Chemotaxis analysis showed that LZIP expression because of the NF-B subfamily is specifically involved in Lkn-1-induced chemotaxis. Our findings suggest that transcription factor NF-B plays an important role in regulation of LZIP expression, and LZIP expression regulates the monocyte cell migration induced by Lkn-1.The human leucine zipper protein LZIP binds to canonical cAMP-responsive elements (CREs) 2 and can activate transcription from CRE-containing reporter genes (1-3). LZIP also binds to the CCAAT enhancer-binding protein element (1). LZIP is known to be involved in the regulation of cell growth by binding to the human host cell factor, which is involved in cell proliferation (4). It has also been suggested that LZIP serves a novel cellular tumor suppressor function that is targeted by the hepatitis C virus core protein (4). We have recently reported that LZIP associates with CC chemokine receptor 1 (CCR1) and up-regulates leukotactin-1 (Lkn-1)-induced chemotactic activity, indicating that LZIP functions as a positive regulator in leukocyte cell migration induced by chemokines (5). However, the exact biological roles and the natural target genes of LZIP remain to be characterized.The human LZIP gene is located on chromosome 9p and consists of nine exons and eight introns. Sequence analysis of the 5Ј-flanking region of the LZIP gene has revealed that LZIP promoter has potential binding sites for transcription factors involved in inflammatory processes and regulation of the immune system, including nuclear factor B (NF-B), nuclear factor of activated T-cells, specific protein-1 (Sp-1), and activator protein-2 (AP-2) (6). However, the regulatory mechanism of the transcriptional activation of LZIP is still obscure.Chemokines are a large family of chemotactic cytokines that provide key signals for traff...
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