Interferon regulatory factor-2 is a transcriptional activator in muscle where It regulates expression of vascular cell adhesion molecule-1 - PubMed (original) (raw)

Interferon regulatory factor-2 is a transcriptional activator in muscle where It regulates expression of vascular cell adhesion molecule-1

T L Jesse et al. J Cell Biol. 1998.

Abstract

Previously, we have suggested that vascular cell adhesion molecule-1 (VCAM-1) and its integrin receptor alpha4beta1 mediate cell-cell interactions important for skeletal myogenesis. Expression of the receptors subsequently subsides in muscle after birth. Here, we examine the mechanism regulating VCAM-1 gene expression in muscle. An enhancer located between the TATA box and the transcriptional start site is responsible for VCAM-1 gene expression in muscle-this element is inactive in endothelial cells where VCAM-1 expression is dependent on nuclear factor kappaB sites and inflammatory cytokines. We identify interferon regulatory factor-2 (IRF-2), a member of the interferon regulatory factor family, as the enhancer-binding transcription factor and show that expression of IRF-2 parallels that of VCAM-1 during mouse skeletal myogenesis. IRF-2 is not dependent upon cytokines for expression or activity, and it has been shown to act as a repressor in other nonmuscle cell types. We show that the basic repressor motif located near the COOH-terminal of IRF-2 is not active in muscle cells, but instead an acidic region in the center of the molecule functions as a transactivating domain. Although IRF-2 and VCAM-1 expression diminishes on adult muscle fiber, they are retained on myogenic stem cells (satellite cells). These satellite cells proliferate and fuse to regenerate muscle fiber after injury or disease. We present evidence that VCAM-1 on satellite cells mediates their interaction with alpha4beta1(+) leukocytes that invade the muscle after injury or disease. We propose that VCAM-1 on endothelium generally recruits leukocytes to muscle after injury, whereas subsequent interaction with VCAM-1 on regenerating muscle cells focuses the invading leukocytes specifically to the sites of regeneration.

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Figures

Figure 1

An element between the TATA box and transcriptional start site controls VCAM-1 expression in skeletal muscle cells. Wild-type and mutant VCAM-1 promoter constructs (20 μg) were transfected into C2C12 myoblasts. Numbers in the name indicate the amount of VCAM-1 gene 5′ flanking region in each construct (−32, 32 bp; −288, 288 bp; −2.1, 2.1 kb). In mutant constructs, the IRF sequence between positions −21 and −5 bp was replaced with the following sequence, where mutated bases are indicated by lowercase letters: 5′-cCgGctcccgcgcTcCg-3′. The results with each construct are representative of at least five different assays done in duplicate, and using different preparations of plasmid DNAs. Mutation of the IRF site always resulted in at least a sevenfold decrease in reporter activity.

Figure 2

The IRF element is necessary for VCAM-1 gene promoter activity in C2C12 myoblasts. Wild-type and mutant −32 VCAMCAT reporter constructs were transfected into C2C12 myoblasts. Black boxes indicate mutated regions in the VCAM-1 gene promoter. The wild-type VCAM-1 sequence between positions −32 and +5 bp is shown above. The sequence of the mutants between positions −23 and +1 bp, where mutated bases are indicated by lowercase letters, are as follows: 5′-cCcggcACTTTCTATTTCACTCCG-3′ (−23 to −18); 5′-GCcCgGctcccgcgcTcCgCTCCG-3′ (−21 to −5); 5′-GCcCgGctcccgTATTTCACTCCG-3′ (−21 to −12); 5′-GCACAGtaTTTCTATTTCACTCCG-3′ (−17 to −16); 5′-GCACAGACggTCTATTTCACTCCG-3′ (−15 to −14); 5′-GCACAGACTTgaTATTTCACTCCG-3′ (−13 to −12); 5′-GCACAGACTcTCTATgTCACTCCG-3′ (−14 and −8); 5′-GCACAGACTTTCTAcgcgcCTCCG-3′ (−9 to −5); 5′-GCACAGAggctcccgTCACTCCG-3′ (−15 to −8); 5′-GCACAGACTTTCccTTTCACTCCG-3′ (−11 to −10); 5′-GCACAGACTTTCTATTTCcCTCCG-3′ (−5); 5′-GCACAGACTTTCTATTTCgaTCCG-3′ (−5 to −4); 5′-GCACAGACTTTCTATTTCACTCgt-3′ (−1 to +1). The results with each construct are representative of at least five independent assays done in duplicate, with multiple preps of plasmid DNAs. There is not a significant difference between −21 to −5, −21 to −12, −15 to −14, −14 and −8, −9 to −5, or −15 to −8. Likewise, there is no significant difference between wild-type, −23 to −18, −17 to −16, −11 to −10, or −1 to +1. In contrast, −5 to −4 and −13 to −12 were reproducibly different from the other two groups.

Figure 3

Expression of IRF proteins. (A) Extracts were prepared from undifferentiated and retinoic acid–treated P19 cells (15 μg), C2C12 myoblasts (15 μg), and embryonic and adult skeletal muscle (30 μg), separated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti–IRF-2 antisera. (B) 15 μg of whole cell extracts prepared from untreated and IFN-γ-treated (3 h, 1000 U/ml) NIH 3T3 and C2C12 cells were separated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti–IRF-1 antisera. NS denotes a nonspecific complex.

Figure 4

IRF-2 binds to the VCAM-1 gene promoter in C2C12 myoblasts. A gel retardation assay is shown using C2C12 nuclear extract with a probe corresponding to positions −32 to +8 bp in the VCAM-1 gene promoter. A 100- or 250-fold molar excess of unlabeled competitor oligonucleotides or 2.5 μl of antisera (Ab) was included in the assays where indicated. HLA indicates a control IRF binding element from the HLA-B7 gene promoter (Johnson and Pober, 1994). NS denotes a nonspecific complex. −5/−21 indicates that the mutant sequence described in Fig. 1 was used as a competitor or a labeled probe. On the right-hand side of the figure, a 250-fold excess of competitor oligonucleotides was used.

Figure 5

A central acidic region of IRF-2 is responsible for transactivation of the VCAM-1 gene promoter. (A) Schematic representation of full-length IRF-2 (FL) activating the VCAM-1 gene promoter, and a dominant-negative IRF-2(160), which contains the DNA binding domain but lacks the acidic transcriptional activation domain, displacing wild-type IRF-2 and thereby inhibiting transcription. (B) Schematic representation of IRF-2 deletion proteins used in transfection studies. Numbers in parenthesis indicate the length in amino acids. (C) C2C12 myoblasts were cotransfected with 10 μg of reporter construct, either −32 VCAMCAT or −2.1 VCAMCAT, and the indicated amount of empty vector (−) or IRF expression vector. (D) C2C12 myoblasts were cotransfected with 10 μg of reporter construct, either −32 VCAMCAT or −32 VCAMCAT-Mut, which contains the mutated sequence described in Fig. 1 (−21/−5). (E) A gel retardation assay showing the binding of full-length IRF-2 and IRF-2 deletion mutants to the VCAM-1 IRF-2 site. CAT assays are all representative of at least five different assays, each done in duplicate with multiple preps of plasmid DNAs.

Figure 5

Figure 6

Expression of VCAM-1 closely follows that of IRF-2 in skeletal muscle differentiation. (A) IRF-2 and VCAM-1 expression follow that of the muscle differentiation marker myosin heavy chain during myogenesis in the mouse. (a) Myosin heavy chain immunostaining in differentiating somites in the mouse at E11. A sagittal section is shown. (b) Double immunostaining of the section in A for IRF-2. (c) Immunostaining of an adjacent section for VCAM-1. Arrows indicate differentiating somites. (B) Expression of IRF-2 precedes expression of VCAM-1 in differentiating mouse skeletal muscle. (a) Low power view (5×) of a section of E15 mouse immunostained for IRF-2. Arrows indicate IRF-2-positive muscle masses. (b) Double immunostaining of the section in a for VCAM-1. (c) A 10× magnification of a section of E15 mouse immunostained for IRF-2. Intense staining is evident only on some muscle masses. (d) High power view of muscle masses from E15 mouse immunostained for myosin heavy chain. (e) The section in e was treated with bis-benzimide and nuclei were viewed by UV illumination. (f) The section in d and e was double immunostained for IRF-2. Note that IRF-2 is only expressed on a subset of the myosin-positive cells in the muscle mass. (g) High power view of another E15 mouse muscle mass immunostained for IRF-2. (h) Bis-benzimide nuclear staining for the section in g. (i) Double immunostaining of the section in g and h for VCAM-1. Note that a very low level of VCAM-1 immunostaining is evident around a small subset of the cells in the muscle mass. Arrows indicate the same location in d–f and g–i, respectively. (C) Expression of VCAM-1 and IRF-2 coincide in mouse skeletal muscle at E18. (a) Low power view of IRF-2 immunostaining of muscle masses in a section of an E18 mouse. R indicates a rib. (b) Bis-benzimide nuclear staining of the section in a. Arrows in a and b indicate the same location. (c) High power view of immunostaining of E18 muscle mass for myosin heavy chain. (d) Bis-benzimide nuclear staining of the section in c. (e) Double immunostaining of the section in c and d for IRF-2. Note that in contrast to E15 (B, d–f), all of the myosin-positive cells in the muscle mass are now IRF-2 positive. Arrows in c–e indicate the same location. (f) High power view of immunostaining of another E18 muscle mass for IRF-2. (g) Bis-benzimide nuclear staining of the section in f. (h) Double immunostaining of the section in f and g for VCAM-1. Note that VCAM-1 is now expressed around the IRF-2-positive cells in the muscle mass. Arrows indicate the same location in f–h. Similar patterns of IRF-2 immunostaining were observed with three different antibodies.

Figure 6

Figure 7

IRF-2 and VCAM-1 are restricted to satellite cells in adult skeletal muscle. (A) Bis-benzimide nuclear staining of a section of mouse adult skeletal muscle. (B) Immunostaining of the section in A for VCAM-1. (C) Double immunostaining of the section in A and B for IRF-2. Arrows indicate IRF-2/VCAM-1-positive cells. (D) High power view of laminin immunostaining of a section of mouse adult skeletal muscle. (E) The section in D was double immunostained for VCAM-1. Note that the VCAM-1-positive cells in the adult muscle are wedged between the basement membrane and the muscle fiber, a location indicative of satellite cells. Arrows in D and E indicate the same location. (F and G) VCAM-1-positive cells in adult skeletal muscle are also positive for the satellite cell marker NCAM. G and F show double immunostaining of a section of adult mouse skeletal muscle for VCAM-1 and NCAM, respectively. Bar, = 25 μm.

Figure 7

Figure 8

VCAM-1 is expressed after muscle injury and during muscle disease in adult mice. The gastrocnemius hindlimb muscle of adult mice was exposed surgically and damaged by cutting. 6 d after surgery, the mice were killed and the region of damaged muscle along with muscle from a corresponding site in the control hindlimb muscle were removed, and frozen sections were prepared. Immunostaining for laminin in control and damaged muscle is shown in A and B, respectively. Arrows in B denote the site of damage. Small, newly forming myotubes are evident in the damaged area (to the right of the arrows). C and D show immunostaining for NCAM and VCAM-1, respectively, in control muscle. E and F show double immunostaining for NCAM and VCAM-1, respectively, in damaged muscle. Arrows indicate the same location in the two panels. Note the presence of groups of NCAM(+)/ VCAM-1(+) small cells that appear to be proliferating satellite cells and larger cells that appear to be the myotubes that form from the satellite cell fusion. G and H show immunostaining for leukocytes (anti–CD45) in normal and damaged muscle, respectively. I and K show immunostaining for VCAM-1 in damaged muscle, whereas J and L show the same sections double immunostained for CD45. Arrows indicate the same location in the panels. Note the close association of the CD45(+) leukocytes and the VCAM-1(+) muscle cells. M and N show immunostaining for CD45 and VCAM-1, respectively, in sections of limb muscle from dystrophin-deficient mice. Results are all representative of at least four separate experiments.

Figure 9

VCAM-1/α4β1 interactions mediate binding of lymphocytes to satellite cells. (A) Photomicrograph of C2C12 satellite cells growing as a monolayer on a tissue culture dish. (B) Photomicrograph showing adhesion of the Ramos T-cell line to monolayers of C2C12 cells. (C) Addition of a control antimyosin heavy chain antibody does not inhibit interaction of Ramos cells with C2C12 cells. (D) Addition of an antibody that specifically blocks the VCAM-1/α4β1 interaction disrupts binding of Ramos cells to the C2C12 cells (see Materials and Methods).

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Interferon regulatory factor-2 is a transcriptional activator in muscle where It regulates expression of vascular cell adhesion molecule-1 - PubMed (original) (raw)