Granulocytic nuclear differentiation of lamin B receptor–deficient mouse EPRO cells

Abstract

Objective-Lamin B receptor (LBR) is an integral protein of the inner nuclear membrane. Recent studies have demonstrated that genetic deficiency of LBR during granulopoiesis results in hypolobulation of the mature neutrophil nucleus, as observed in human Pelger-Huët anomaly (PHA) and mouse ichthyosis (ic). In this study we have utilized differentiated promyelocytes (EPRO cells) that were derived from the bone marrow of homozygous and heterozygous ichthyosis mice to examine changes to the expression of nuclear envelope proteins and heterochromatin structure that result from deficient LBR expression.Materials and Methods-Wildtype (+/+), heterozygous (+/ic) and homozygous (ic/ic) granulocytic forms of EPRO cells were analyzed for the expression of multiple lamins and inner nuclear envelope proteins by immunostaining and immunoblotting techniques. The heterochromatin architecture was also examined by immunostaining for histone lysine methylation.Results-Wildtype (+/+) and heterozygous (+/ic) granulocytic forms revealed ring-shaped nuclei and contained LBR within the nuclear envelope; ic/ic granulocytes exhibited smaller ovoid nuclei devoid of LBR. The pericentric heterochromatin of undifferentiated and granulocytic ic/ic cells was condensed into larger spots and shifted away from the nuclear envelope, compared to +/+ and +/ic cell forms. Lamin A/C, which is normally not present in mature granulocytes, was significantly elevated in LBR-deficient EPRO cells.Conclusions-Our observations suggest roles for LBR during granulopoiesis which may involve augmenting nuclear membrane growth, facilitating compartmentalization of heterochromatin and promoting down-regulation of lamin A/C expression. Category Granulopoiesis

Mouse neutrophils lacking lamin B-receptor expression exhibit aberrant development and lack critical functional responses

“…These results support the conclusion that the ic/ic cells are homozygous for a hypomorphic allele that yields very low Lbr transcript expression. Furthermore, absent protein expression in these cells is reported in the subsequent studies by Zwerger et al [22]. Interestingly, the expression of genes that encode either the secondary granule protein lactoferrin or the neutrophil-specific transcriptional regulator C/ EBPε was not affected by the ichthyosis mutation in ATRA-induced EPRO-ic/ic cells (Fig.…”

“…Thus our findings that the lack of LBR expression in EPRO-ic/ic cells inhibits distinct neutrophil functions and the expression of a single component of the NADPH oxidase are not unusual and may implicate the function of nuclear membrane components in transcriptional regulation. Interestingly, our subsequent study presents evidence that the lack of LBR expression in EPRO-ic/ic cells correlates with increased expression of lamin A/C [22].…”

“…We demonstrate that the defective respiratory burst is likely due in part to a loss of gp91 phox expression, suggesting that deficient LBR expression may also affect transcriptional regulation of this critical oxidase. In the article following this study, the Lbr mutation in ic/ic cells is shown to affect the expression of multiple proteins integral to the neutrophil nucleus (including LBR), and also alter the distribution of heterochromatin markers [22]. Together, our data provide novel evidence that LBR expression is essential not only for neutrophil nuclear lobulation but also for normal neutrophil functional responses and neutrophil-specific gene expression.…”

An epichromatin epitope: Persistence in the cell cycle and conservation in evolution

“…[50][51][52] To cite one example, pericentromeric heterochromatin regions of mouse granulocytes frequently cluster adjacent to the nuclear envelope making nodules into the cytoplasm. 53 In this situation, it is evident that the nuclear envelope integral membrane protein LBR is necessary for the heterochromatin to exhibit a peripheral localization. In another remarkable variation of interphase nuclear architecture, heterochromatin in the retinal rod cells of nocturnal (but not diurnal) mammals is "inverted" (clustered) into the middle of nuclei, apparently to function in channeling the light.…”

Dosage effect of zero to three functionalLBR-genes in vivo and in vitro

“…15,16 If progenitor EML cells are generated from these mice and induced to mature neutrophils, the cells also exhibit nuclear hyposegmentation. 17,18 The typical nuclear structure of human neutrophils is an evolutionarily evolved and conserved characteristic. Interspecies comparison of nuclear morphology in neutrophils revealed ovoid nuclei in most non-vertebrates, followed by rodlike or bilobed nuclei in fish and birds, and multi-segmented nuclei in neutrophils of most mammals.…”