Jühlen et al. Cell Div (2018) 13:8 https://doi.org/10.1186/s13008-018-0041-5 Cell Division RESEARCH Open Access Triple A patient cells sufering from mitotic defects fail to localize PGRMC1 to mitotic kinetochore fbers Ramona Jühlen1,2* , Dana Landgraf1, Angela Huebner1 and Katrin Koehler1* Abstract Background: Membrane-associated progesterone receptors are restricted to the endoplasmic reticulum and are shown to regulate the activity of cytochrome P450 enzymes which are involved in steroidogenesis or drug detoxifca- tion. PGRMC1 and PGRMC2 belong to the membrane-associated progesterone receptor family and are of interest due to their suspected role during cell cycle. PGRMC1 and PGRMC2 are thought to bind to each other; thereby suppress- ing entry into mitosis. We could previously report that PGRMC2 interacts with the nucleoporin ALADIN which when mutated results in the autosomal recessive disorder triple A syndrome. ALADIN is a novel regulator of mitotic control- ler Aurora kinase A and depletion of this nucleoporin leads to microtubule instability. Results: In the current study, we present that proliferation is decreased when ALADIN, PGRMC1 or PGRMC2 are over- expressed. Furthermore, we fnd that depletion of ALADIN results in mislocalization of Aurora kinase A and PGRMC1 in metaphase cells. Additionally, PGRMC2 is over-expressed in triple A patient fbroblasts. Conclusion: Our results emphasize the possibility that loss of the regulatory association between ALADIN and PGRMC2 gives rise to a depletion of PGRMC1 at kinetochore fbers. This observation may explain part of the symp- toms seen in triple A syndrome patients. Keywords: ALADIN, Kinetochore fbers, Mitosis, Nuclear pore complex, PGRMC1, PGRMC2, Triple A syndrome Background was published in 2015 [7]. In cooperation with Carvalhal ALADIN is a scafold nucleoporin (NUP) anchored et al. we proposed ALADIN as novel regulator of mitotic within the nuclear pore complex by the transmembrane kinase Aurora kinase A and showed that depletion of the NUP NDC1 [1, 2]. ALADIN seems to be involved in nucleoporin resulted in impaired mitotic spindle assem- building the structural scafold backbone of the nuclear bly and chromosomal alignment at the metaphase plate pore complex [3]. Over the last years it has been shown [7]. Furthermore, we could recently document that ALA- that nuclear pore complexes and its NUPs have fun- DIN is necessary for murine oocyte maturation and for damental functions beyond nucleocytoplasmic trans- specifc stages during meiosis [8]. port and control cellular dysfunction in a variety of Mutations in the human AAAS gene, coding for the cellular pathways, especially during mitosis [4–6]. Te protein ALADIN (alacrima-achalasia-adrenal insuf- frst report that ALADIN plays a role during cell division fciency neurologic disorder), lead to the autosomal recessive disorder named triple A syndrome [9, 10]. Triple A patients present with three distinct symptoms: *Correspondence: [email protected]; katrin. absent adrenal glucocorticoid and mineralocorticoid koehler@uniklinikum‑dresden.de synthesis (adrenal insufciency), impaired movement 1 Klinik und Poliklinik für Kinder‑ und Jugendmedizin, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, of the stomach cardia (achalasia) and loss of tear pro- 01307 Dresden, Germany duction (alacrima) [11]. Tese symptoms are highly 2 Present Address: Institute for Molecular Biology and Medicine, Université heterogeneous and are accompanied by progressive Libre de Bruxelles, 6041 Charleroi, Belgium © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jühlen et al. Cell Div (2018) 13:8 Page 2 of 14 impairments of the central, peripheral or autonomous (p58) [28]. PGRMC1 and PGRMC2 are reported to inter- nervous system [11]. Most mutations in AAAS result act and furthermore, to bind to each other during meta- in a mis-localization of ALADIN to the cytoplasm [12, phase, thereby suppressing entry into cell cycle [27]. 13]. Interestingly, in a large scale interactome mapping of Previously, we identifed PGRMC2 as novel interac- the centrosome-cilium interface the nucleoporin ALA- tor for the nucleoporin ALADIN and provided new DIN and both membrane-associated progesterone recep- insights into the molecular function of the nucleoporin tors PGRMC1 and PGRMC2 have been identifed to in the pathogenesis of triple A syndrome [14]. PGRMC2 localize to the cilium transition zone [31, 32]. Te centro- belongs to the group of membrane-associated proges- some is an important regulator of cell cycle progression terone receptors. Tese receptors mainly localize to the and mitotic spindle assembly [33]. Furthermore, ALA- endoplasmic reticulum (ER) and are thought to regu- DIN is strongly dephosphorylated during mitotic exit, late the activity of microsomal cytochrome (CYP) P450 whereas PGRMC1 and PGRMC2 are phosphorylated enzymes which are involved in steroidogenesis or drug during early mitotic exit [34, 35]. Obviously, ALADIN, detoxifcation [15]. Te frst identifed membrane- PGRMC1 and PGRMC2 seem to have critical roles for associated progesterone receptor, PGRMC1, gained the formation and function of the mitotic spindle appara- wide-spread attention due to its several implications in tus in somatic cells. cancerogenesis [16–19]. Te mixed-function oxidase Here, we report that proliferation in human adrenal system of CYP P450 enzymes requires a donor trans- cells is decreased after increased expression of ALADIN, ferring electrons from NADPH to reduce the enzyme’s PGRMC1 or PGRMC2. We show that PGRMC1 local- prosthetic heme group [20]. PGRMC1 and PGRMC2 izes to the microtubule kinetochore-fbers in metaphase contain a CYP b5-similar heme-binding domain which and to the midbody in telophase of human adrenal cells makes them possible electron donors for CYP P450 and fbroblasts. Further, we present that PGRMC1 and enzymes. [19]. Indeed, PGRMC1 forms stable protein– Aurora kinase A are mislocalized in metaphase triple protein complexes with CYP51A1, CYP7A1, CYP21A2 A patient fbroblasts. We observed that patient fbro- and CYP3A4 [21]. Additionally, PGRMC1 is able to acti- blasts present with increased expression of PGRMC2 vate CYP19 aromatase [22]. PGRMC1 is shown to physi- and we hypothesize that a depletion of ALADIN in these ologically afect cholesterol/steroid biosynthesis and cells leads to a dysregulation of PGRMC2 and displaces metabolism [19]. It is known that PGRMC2 has similar PGRMC1 at the metaphase spindle. interaction potential, alters activity of CYP3A4 as pos- sible electron donor, and binds CYP21A2 [23, 24]. Most Results recently, both PGRMC1 and PGRMC2 were identifed Adrenal cell proliferation is decreased as putative interacting partners of ferrochelatase, an upon over‑expression of ALADIN, PGRMC1 or PGRMC2 enzyme catalyzing the terminal step in the heme biosyn- and down‑regulation of ALADIN thetic pathway, thereby possibly controlling heme release It is well documented that triple A patient fbroblasts as chaperone or sensor [25]. Interaction of ALADIN with show decreased cellular proliferation and doubling PGRMC2 at the perinuclear ER could infuence CYP time [36]. To test whether depletion of ALADIN also P450 enzyme activity through electron transfer from results in decreased cellular proliferation in adrenal NADPH and/or control heme synthesis. In triple A syn- cells we used inducible adrenocortical NCI-H295R1- drome, altered CYP P450 enzyme activity would consec- TR cells stably expressing AAAS shRNA (AAAS utively contribute to adrenal atrophy [14]. knock-down) and monitored cellular proliferation Human PGRMC1 and PGRMC2 share 67% of their using live cell imaging for at least 65 h. AAAS knock- protein sequence [15, 26]. Defciency of either PGRMC1 down resulted in decreased proliferation in adrenal or PGRMC2 decreases the anti-apoptotic and anti- cells (growth constant k (slope of linear regression mitotic action of progesterone [27]. Additionally, line) = 0.044) compared to control cells expressing a increased expression of PGRMC1 or PGRMC2 inhibits scrambled shRNA (k = 0.062) (wild-type cells: k = 0.2) entry into cell cycle [27, 28]. On the one hand, PGRMC1 (Fig. 1a). Surprisingly, in live cell imaging stable over- is distributed with β- and γ-tubulin to the mitotic bipo- expression of N-terminal-GFP-tagged ALADIN in lar spindle and spindle poles in metaphase cells and on adrenocortical NCI-H295R cells also impaired cellu- the other hand, with Aurora kinase B in meiotic cells lar proliferation (k = 0.082) compared to over-expres- [29, 30]. Furthermore, PGRMC1 is thought to regulate sion of GFP alone in these cells (k = 0.305) (wild-type microtubule stability [28]. PGRMC2 is shown to local- cells: k = 0.16) (Fig. 1b). Furthermore, during live cell ize to the mitotic spindles in metaphase and anaphase imaging we observed that transient over-expression of cells and shall interact with cyclin-dependent kinase 11B C-terminal-GFP-tagged PGRMC2 in adrenocortical Jühlen et al. Cell