Abstract
The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actin-associated 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 recombinase-induced 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.
Highlights
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]
The present study reports for the first time the high level expression of h2-calponin in the monocyte-granulocyte lineage of myeloid cells with an up-regulation during monocyte to macrophage differentiation
While the h2-calponin-free macrophages in vitro using fluorescent latex beads. nin expression is dependent on the differentiation of granulo
Summary
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]. We previously demonstrated that h2-calponin is expressed at higher levels in developing and remodeling smooth muscles and its overexpression inhibited the rate of cell proliferation [18]. The function of h2-calponin in regulating the actin cytoskeleton of non-muscle cells suggests its role in multiple cellular activities. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed through embryonic stem (ES) cell gene targeting [19]. The insertion of the neoR selection cassette into intron 2 of the Cnn gene resulted in a significant knockdown of h2-calponin expression. The lack of h2-calponin significantly increased phagocytotic activity, suggesting a novel mechanism to regulate macrophage function
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