Definitive evidence using enucleated cytoplasts for a nongenomic basis for the cystic change in endoplasmic reticulum structure caused by STAT5a/b siRNAs

Abstract

STAT5a/b species are well known as transcription factors that regulate nuclear gene expression. In a novel line of research in human pulmonary arterial endothelial cells (HPAECs), we previously observed that STAT5a associated with the Golgi apparatus and that siRNA-mediated knockdown of STAT5a/b led to the rapid development of a dramatic cystic change in the endoplasmic reticulum (ER) characterized by deposition along cyst membranes and tubule-to-cyst boundaries of the proteins reticulon-4 (RTN4; also called Nogo-B) and the ER-resident GTPase atlastin-3 (ATL3) and Golgi fragmentation. We now report that STAT5a can be observed in ER sheets in digitonin-permeabilized HPAECs and that anti-STAT5a cross- immunopanned ATL3 but not RTN4. Moreover, there was marked accumulation of the 63-kDa cytoskeleton-linking membrane protein and ER-spacer CLIMP63 (also called cytoskeleton-associated protein 4, CKAP4) and KDEL-mCherry within the cysts. That the STAT5a/b-siRNA-induced cystic ER phenotype developed in the presence of the transcription inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) had suggested that the mechanism was independent of the transcription factor functions of STAT5a/b, i.e., was "nongenomic." We have now definitively tested the requirement for the nucleus in eliciting the STAT5a/b-siRNA-induced cystic ER phenotype. Enucleated HPAEC cytoplasts were prepared using adherent 35-mm cultures using the cytochalasin B-centrifugation method (typically yielding 65-75% enucleation). STAT5a/b siRNAs readily elicited the cystic ER phenotype including the marked luminal accumulation of CLIMP63 and Golgi fragmentation in the recovered HPAEC cytoplasts demonstrably lacking a nucleus. These studies provide unequivocal evidence using enucleated cytoplasts for a nongenomic mechanism(s) underlying the cystic change in ER structure elicited by STAT5a/b knockdown.

MxA Is a Novel Regulator of Endosome-Associated Transcriptional Signaling by Bone Morphogenetic Proteins 4 and 9 (BMP4 and BMP9)

“…These cells were grown in DMEM supplemented with 10% v/v fetal bovine serum (FBS). HEK293T cells were seeded into 6-well, 24-well plates or 35mm-dishes coated with fibronectin, collagen and bovine serum albumin (respectively 1 μg/ml, 30 μg/ml and 10 μg/ml in coating medium) [41,42,43]. Primary human pulmonary arterial endothelial cells (HPAEC) were purchased from Clonetics (San Diego, CA).…”

“…Primary human pulmonary arterial endothelial cells (HPAEC) were purchased from Clonetics (San Diego, CA). These were seeded into T-25, T-75 or 6-well plates coated with fibronectin, collagen and bovine serum albumin (respectively 1 μg/ml, 30 μg/ml and 10 μg/ml in coating medium) [41,42,43]. HPAECs were grown in Medium 200 supplemented with low serum growth supplement LSGS (Cascade Biologics, Carlsbad, CA) and were used between passages 4 and 10 [44].…”

“…Whole cell extracts, as well as cytoplasmic and nuclear extracts using hypotonic swelling and Dounce breakage, were prepared from HEK293T cells as described previously [31,41,42] and citations therein). Western blotting was carried out using 8, 10 or 12% SDS-polyacrylamide gels under reducing denaturing conditions as per procedures and protocols provided by Cell Signaling Technology Inc. and ECL detection kit purchased from Thermo Scientific (34077) (Rockford, IL) and Michigan Diagnostics (FWPD02-25) (Royal Oak, MI).…”

Subcellular Mechanisms in Pulmonary Arterial Hypertension: Combinatorial Modalities that Inhibit Anterograde Trafficking and Cause Bone Morphogenetic Protein Receptor Type 2 Mislocalization

“…19,20 The cells were imaged using a Zeiss AxioImager M2 motorized microscopy system with Zeiss W N-Achroplan 40×/NA0.75 or Zeiss EC Plan-Neofluor 100×/NA1.3 oil objectives equipped with a high-resolution RGB HRc AxioCam camera and AxioVision software (ver. 4.8.1) in 1,388 × 1,040-pixel high-speed color-capture mode.…”

“…We discovered this "nongenomic" membrane trafficking function in the cytoplasm 19,20 for the "transcription factors" STAT5a and STAT5b. [21][22][23][24] STAT5a/b proteins associated with structural components of the ER/Golgi and their downregulation led to a characteristic cystic ER/Golgi fragmentation phenotype in primary human pulmonary arterial endothelial cells (HPAECs) and human pulmonary arterial smooth muscle cells (HPASMCs).…”