Lymphohematopoietic licence: Sterol C-14 reductase activity of lamin B receptor (Lbr) is essential for neutrophil differentiation.

ABSTRACT

Bertie Wooster, PG Wodehouse’s fictional character, proudly fancied himself a writer for having once contributed an article (‘What the Well-Dressed Man Is Wearing’) to Milady’s Boudoir, his Aunt Dahlia’s weekly magazine for women. My conceit of expertise in vertebrate lamin B receptor (Lbr) research is slightly less dubious. I co-authored two papers (Papavinasasundaram and Kasbekar 1994 and Prakash et al. 1999) that established that the C-terminal two-thirds of Lbr has sterol Δ14,15 reductase activity. An interloping article (Silve et al. 1998) reached pretty much the same conclusion (and to my chagrin, garnered the lion’s share of the citations). So the recent demonstration by Peter Gaines and coworkers that the Lbr sterol reductase regulates differentiation of neutrophils (Subramanian et al. 2012) filled me with proprietary pride, especially since neutrophils are the most abundant white blood cells in circulation and present the critical first line of defence against infectious microbes. Lbr is an integral protein of the vertebrate nuclear envelope inner membrane. Its N-terminal ~200 residues are hydrophilic, bind to B-type lamins, DNA and HP1-type chromatin proteins, and provide a substrate for p34cdc2, a key mitotic protein kinase. The ~420 residue hydrophobic, membrane-spanning, C-terminal domain (CTD) with sterol reductase activity anchors the nucleoplasmic domain to the inner nuclear membrane. Mutations in human LBR cause Pelger-Huët anomaly (PHA), a benign dominant disorder characterized by hyposegmentation of the neutrophil nucleus (Hoffmann et al. 2002). A spontaneously aborted fetus with Greenberg/HEM dysplasia was homozygous for LBR mutations, and peripheral blood neutrophils from the fetus’ mother displayed PHA (Waterham et al. 2003). Greenberg/HEM dysplasia and PHA reflect the pleiotropism of LBR mutations. In mouse, the Lbr gene is defined by the ichthyosis (ic) mutations (Shultz et al. 2003). Neutrophils from ic/ic mice display bilobed or ovoid nuclei typical of PHA. Additionally, ic/ic homozyogotes exhibit sparse hair, decreased body size and occasionally hydrocephalus and syndactyly. It was of interest to understand the functional significance of the Lbr protein’s sterol reductase activity, especially since KO mice for another locus, Tm7sf2, that encodes SR-1, a 418 residue protein with 58% identity with the Lbr CTD and possessing sterol C−14 reductase activity, do not display an observable phenotype (Bennati et al. 2006, 2008). First, a quick flashback to another 1994 paper Tsai et al. (1994) had shown that transduction of normal mouse bone marrow cells with a retroviral vector harbouring a dominant-negative retinoic acid receptor (RARα403) could reproducibly immortalize lymphohematopoietic progenitors as stem-cell-factor-dependent clonal lines, designated as EML cells for their ability to subsequently undergo erythroid, myeloid and lymphoid differentiation in vitro. A 3-day treatment of EML cells with stem cell factor, IL-3, and high concentrations of all-trans retinoic acid, and then washing and switching them into GM-CSF, induced their differentiation into promyelocytes, designated as EPRO cells (EML-derived promyelocytes), that can be maintained in GM-CSF. Treatment of EPRO cells with high concentrations of retinoic acid in the presence of GM-CSF induced them to terminally differentiate into mature neutrophils with characteristic nuclear lobulation and respiratory burst response phenotypes. Several years later, Gaines et al. (2008) generated EML- and EPRO-like cells frombonemarrow of a C57BL/6J-Lbric-J/Lbric-J (ic/ic) mouse and a normal (+/ic) littermate, and found that neutrophils derived from EPRO-ic/ic cells exhibited nuclear hypolobulation identical to that seen in ichthyosis mice and displayed a deficient respiratory burst, whereas those from.