Role of H2-calponin in Regulating Macrophage Motility and Phagocytosis

Abstract

The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actinassociated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neo R cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neo R cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinaseinduced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions.Leukocytes are mobile cells and their actin cytoskeleton plays a central role in the locomotion, transmigration, and phagocytosis. These activities are essential for the function of myeloid cells, including neutrophils, monocytes, and macrophages, in defensive and autoimmune responses (1). Despite the significant biological and medical importance, the regulation of actin cytoskeleton in myeloid cells is not well understood.Calponin is an actin filament-associated protein of 34 -37 kDa (292-330 amino acids) found in smooth muscle (2) as well as non-muscle cells (3,4). Through high affinity binding to F-actin, calponin inhibits the actin-activated myosin MgATPase (5-8) and motor activity (9 -11). The association of calponin with actin filaments and its regulatory function have led to a model wherein calponin may represent a thin filament regulatory mechanism modulating smooth muscle contraction (12).Three isoforms of calponin (h1, h2, and acidic) have been identified in higher vertebrates as the products of three homologous genes (13-17). The three isoforms of calponin have distinct theoretical isoelectric points (pI values): H1-calponin is basic (pI 9.4), h2-calponin is near neutral (pI 7.5), whereas the acidic calponin has a lower pI of 5.2. H1-calponin is the predominant isoform specifically expressed in differentiated smooth muscle cells and its role in regulating smooth muscle contractility (9) is a focus of ongoing research. The majority of previous structural and functional studies of calponin were obtained from chicken gizzard calponin that is equivalent to the mammalian h1-calponin. The acidic c...

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