Lamin B Receptor Regulates the Growth and Maturation of Myeloid Progenitors via its Sterol Reductase Domain: Implications for Cholesterol Biosynthesis in Regulating Myelopoiesis

Abstract

Lamin B receptor (LBR) is a bifunctional nuclear membrane protein with N-terminal lamin B and chromatin binding domains plus a C-terminal sterol Δ14 reductase domain. LBR expression increases during neutrophil differentiation and deficient expression disrupts neutrophil nuclear lobulation characteristic of Pelger-Huët anomaly. Thus LBR plays a critical role in regulating myeloid differentiation, but how the two functional domains of LBR support this role is currently unclear. We previously identified abnormal proliferation and deficient functional maturation of promyelocytes (EPRO cells) derived from EML-ic/ic cells, a myeloid model of ichthyosis (ic) bone marrow that lacks Lbr expression. Here we provide new evidence that cholesterol biosynthesis is important to myeloid cell growth and is supported by the sterol reductase domain of Lbr. Cholesterol biosynthesis inhibitors caused growth inhibition of EML cells that increased in EPRO cells, whereas cells lacking Lbr exhibited complete growth arrest at both stages. Lipid production increased during wild-type neutrophil maturation, but ic/ic cells exhibited deficient levels of lipid and cholesterol production. Ectopic expression of a full length Lbr in EML-ic/ic cells rescued both nuclear lobulation and growth arrest in cholesterol starvation conditions. Lipid production also was rescued, and a deficient respiratory burst was corrected. Expression of just the C-terminal sterol reductase domain of Lbr in ic/ic cells also improved each of these phenotypes. Our data support the conclusion that the sterol Δ14 reductase domain of LBR plays a critical role in cholesterol biosynthesis, and that this process is essential to both myeloid cell growth and functional maturation.

Cooperative Activity of GABP with PU.1 or C/EBPε Regulates Lamin B Receptor Gene Expression, Implicating Their Roles in Granulocyte Nuclear Maturation

“…Further studies demonstrated that increases in Lbr expression during neutrophil differentiation of these EML/EPRO-like cells coincided with increased expression of both C/EBPε and C/EBPε-regulated neutrophil genes, including those encoding murine neutrophil collagenase and neutrophil gelatinase (20). We therefore investigated whether C/EBPε is required for Lbr transcription.…”

“…To determine in vivo if either GABPα or PU.1 occupies either Ets binding site, or if C/EBPε interacts with the proximal C/EBP binding site, ChIP was performed using extracts from the original EML and EPRO cells, which exhibit varying levels of Lbr (20) and each transcription factor during neutrophil differentiation (Fig. 2A, 2B).…”

“…The different responses could also be due to unique structural features between mouse vs. human neutrophil nuclei; the deformability of mice neutrophil nuclei may be more sensitive to the levels of Lbr expression and therefore loss of Lbr hinders their ability to navigate micron-scale constrictions. Increased expression of Lbr also may be critical to its functions as a C-14 sterol reductase, a property that must increase in order to support demands for cholesterol production required for innate immune functions, including activation of NADPH oxidase complexes dependent on membrane lipid rafts (20). Thus, LBR expression levels must be maintained at certain basal levels early in development to support both cholesterol biosynthesis and physical properties of the nucleus, which is supported by GABP transcriptional activity.…”

“…Equal amounts of the protein extracts (~10 μg) were electrophoresed on NuPAGE 4–12% Bis-Tris gradient gels (Novex, Carlsbad, CA) and then electro-blotted onto Immobilon PVDF membranes (EMD Millipore, Billerica, MA). Membranes were probed with the following Abs and dilutions: GABPα (1:1000; SC-22810), PU.1 (1:1000; SC-352), C/EBPε (1:1000; SC-158), and β-actin (1:1000) (all from Santa Cruz Biotechnology, Dallas, TX), LBR from guinea pig serum [kindly provided by Prof. Harald Herrmann, German Cancer Research Institute, Heidelberg, Germany and previously described (20)], or tubulin (1:1500; Sigma-Aldrich). HRP-conjugated secondary Ab (1:2000, Sigma-Aldrich) was used against primary Abs, and chemiluminescence was detected using Immobilon Western HRP substrate solution (EMD Millipore).…”

“…Importantly, use of the dominant-negative RARα403 expression system allows for the generation of EML-like myeloid cell lines from bone marrow of mice with specific genetic defects, such as deficiency of Lbr or C/EBPε (15, 18). Moreover, the neutrophils derived from EML-like cells generated with this technique express both primary and secondary granule proteins, and exhibit upregulation of transcription factors well-known to regulate neutrophil differentiation (19, 20). …”