A mutated retinoic acid receptor-alpha exhibiting dominant-negative activity alters the lineage development of a multipotent hematopoietic cell line.

Abstract

The retinoic acid receptor (RARot) is expressed in virtually all hematopoietic lineages, but the role of this transcription factor in regulating the growth and differentiation of hematopoietic progenitors is unknown. We have constructed a mutant RARer that both exhibits dominant-negative activity against the normal RARer in transient expression assays in mouse fibroblasts and inhibits retinoic acid-induced neutrophilic differentiation of the HL-60 human promyelocytic leukemia cell line. When this dominant-negative RARa construct is introduced into the multipotent intedeukin-3-dependent FDCP mix A4 murine hematopoietic cell line, there is a rapid switch from spontaneous neutrophil/monocyte differentiation to basophil/mast cell development. Thus, in this multipotent hemopoietic cell line the normal RARa transcription factor and/or related molecules appear to promote the differentiation of neutrophils and monocytes but suppress the development of basophils/mast cells.

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

“…To express a dominant negative form of retinoic acid receptor α (RARα), cells were spinfected for 2 hours with supernatants derived from a stable GP+E86 producer line infected with the LRARα403SN vector (a kind gift of Archibald Perkins, Yale University, New Haven) plus polybrene (4 µg/ml; Sigma, St. Louis , MO) [24]. Following a two week exposure to G418 (0.4 mg/mL, Invitrogen, Carlsbad, CA), EML-like cells emerged after approximately 1 month; thereafter, cells were routinely maintained in Iscove's Modified Dulbecco Medium (IMDM, Invitrogen) supplemented with 20% horse serum (HS) and 15% BHK/MKL-conditioned medium (as a source of SCF).…”

Diverse Functions of Retinoic Acid in Brain Vascular Development

“…Sox17 is a transcription factor that is required for vascular development and its expression is regulated by endothelial WNT signaling in the postnatal CNS vasculature (Ye et al, 2009;. We tested whether the latter was also the case for the fetal brain vasculature using mice with EC conditional knock-down of the WNT signaling component ␤-catenin (PdgfbiCre;Ctnnb1-flox).…”

“…To begin to test this idea, we developed a mouse model in which RA signaling is specifically disrupted in brain ECs using an inducible EC-specific CreER T2 line (Pdgfbi-CreER T2 , referred to here as PdgfbiCre; Claxton et al, 2008) and a conditional, dominantnegative version of RAR␣ allele located in the ROSA26R locus (dnRAR403-flox) (Rosselot et al, 2010). DnRAR␣403 is a truncation mutant of the human RAR␣ that can bind to endogenous RARs, but when expressed in a cell, disrupts endogenous RA signaling activity (Tsai et al, 1992;Damm et al, 1993). To investigate the effect of disrupted endothelial RA signaling on prenatal brain vascular development, pregnant females were injected with tamoxifen at E9 and E10 to induce Cre-mediated expression of dnRAR␣403 in ECs and fetuses were collected at E14.5, E16.5, and E18.5 (Fig.…”

Acute leukemia with promyelocytic features in PML/RARα transgenic mice

“…All these observations taken together would suggest that the deregulation of the PML pathway plays a crucial role in promyelocytic leukemogenesis and that the disruption of this activity by PML͞ RAR␣ could result in the expansion of the APL blasts. On the other hand, the possible role of RAR␣ in the control of myeloid terminal differentiation (24)(25)(26) suggests that the concomitant interference of PML͞RAR␣ with RAR␣͞RXR␣ function could lead to the block of maturation at the promyelocytic stage observed in APL. Indeed, the PML͞RAR␣ protein is capable of blocking the terminal differentiation of myeloid precursor cells such as U937, HL60, or K562 induced by certain stimuli, including vitamin D 3 and transforming growth factor type ␤.…”