The Lamin B Receptor Provides Sterol Reductase Activities Critical to Neutrophil Development: Implications for Cholesterol Biosynthesis In Regulating Myelopoiesis

Abstract 389

The lamin B receptor (LBR) is a unique nuclear envelope protein whose expression is critical to the correct morphologic maturation of differentiating neutrophils. Heterozygous mutations of LBR in humans cause Pelger-Huët anomaly (PHA), a disorder characterized by hypolobulated neutrophil nuclei. Homozygous mutations of LBR cause the lethal disorder HEM/Greenberg skeletal dysplasia in humans and the ichthyosis (ic) phenotype in mice, both of which are associated with severe skeletal defects and severely hypolobulated, or mononuclear neutrophils. Why complete loss of LBR expression causes neutrophil hypolobulation and severe developmental defects remains unclear. The N-terminal domain of LBR, which resides in the nucleoplasm, binds to B-type lamins and chromatin, which is thought to facilitate nuclear envelope reformation at the end of mitosis. The C-terminal domain contains 8 membrane-spanning segments and is homologous to the sterol Δ¹⁴ reductase TM7SF2. Thus phenotypes caused by loss of LBR expression have been considered as laminopathies, i.e. due to disruptions to the functions of lamin B, or due to a disruption in cholesterol biosynthesis. Cholesterol plays a vital role in the production of lipid-enriched microdomains termed lipid rafts, which facilitate the assembly of membrane proteins important for neutrophil adhesion, chemotaxis and the respiratory burst. Furthermore, lipid depravation inhibits the proliferation of myeloblastic HL-60 cells, indicating that cholesterol levels are important to myeloid cell growth. We generated a novel EML progenitor line from the bone marrow of a homozygous ic mouse, termed EML-ic/ic cells, which can be induced to promyelocytic EPRO-ic/ic cells and then into mature neutrophils that display severe PHA identical to those found in ichthyosis mice. To further investigate the importance of cholesterol biosynthesis to neutrophil growth and maturation, we analyzed wild-type EML cells for growth defects when cultured in lipid-deficient serum (LPDS) or when treated with molecules that either deplete cellular cholesterol levels (lovastatin) or sequester membrane cholesterol (methyl-β-cyclodextrin, MβCD). Growth of EML cells was modestly inhibited in all three conditions, but growth of EPRO cells was severely affected. Lipid levels produced by the cells increased as differentiation progressed, suggesting that myeloid progenitors exhibit increased demands for cholesterol as they mature into terminally differentiated neutrophils. By comparison, growth of EML- or EPRO-ic/ic cells was dramatically inhibited when cultured in LPDS, lovastatin or MβCD. Lipid production also was compromised in the ic/ic cells at all stages of development. Both of these defects displayed by EML-ic/ic cells were significantly improved by ectopic expression of a wild-type Lbr. Expression of mutant forms of Lbr that contained the C-terminal domain homologous to TM7SF2, but that lacked binding domains for lamin B or chromatin, also improved proliferation in cholesterol depleted conditions and increased lipid production. Interestingly, while expression of the wild-type Lbr restored nuclear lobulation to the vast majority of ic/ic neutrophils, the mutant forms also were able to rescue lobulation in numerous ic/ic cells. The previously identified respiratory burst defect produced by ic/ic neutrophils also was partly reversed by both wild-type and mutant forms of Lbr. Finally, a Northern analysis demonstrated that neutrophil differentiation caused increased Lbr expression whereas Tm7sf2 expression did not change. In contrast, sterol depravation activated Tm7sf2 expression but did not affect Lbr expression. Together these data indicate that cholesterol biosynthesis is important to the growth and function of neutrophils, that demands for cholesterol increase as neutrophils mature, and that increased Lbr expression during neutrophil differentiation functions to meet these demands. In addition, C-terminal domains of Lbr can support the roles of Lbr in mediating neutrophil survival, morphologic maturation and functional responses. Our data provide new insight into the complex roles that cholesterol play in myelopoiesis, and may be important to understanding the effects of dysfunctional cholesterol biosynthesis to innate immunity provided by neutrophils.