The Regulation of MS-KIF18A Expression and Cross Talk with Estrogen Receptor

This study provides a novel view on the interactions between the MS-KIF18A, a kinesin protein, and estrogen receptor alpha (ERα) which were studied in vivo and in vitro. Additionally, the regulation of MS-KIF18A expression by estrogen was investigated at the gene and protein levels. An association between recombinant proteins; ERα and MS-KIF18A was demonstrated in vitro in a pull down assay. Such interactions were proven also for endogenous proteins in MBA-15 cells were detected prominently in the cytoplasm and are up-regulated by estrogen. Additionally, an association between these proteins and the transcription factor NF-κB was identified. MS-KIF18A mRNA expression was measured in vivo in relation to age and estrogen level in mice and rats models. A decrease in MS-KIF18A mRNA level was measured in old and in OVX-estrogen depleted rats as compared to young animals. The low MS-KIF18A mRNA expression in OVX rats was restored by estrogen treatment. We studied the regulation of MS-KIF18A transcription by estrogen using the luciferase reporter gene and chromatin immuno-percipitation (ChIP) assays. The luciferase reporter gene assay demonstrated an increase in MS-KIF18A promoter activity in response to 10−8 M estrogen and 10−7M ICI-182,780. Complimentary, the ChIP assay quantified the binding of ERα and pcJun to the MS-KIF18A promoter that was enhanced in cells treated by estrogen and ICI-182,780. In addition, cells treated by estrogen expressed higher levels of MS-KIF18A mRNA and protein and the protein turnover in MBA-15 cells was accelerated. Presented data demonstrated that ERα is a defined cargo of MS-KIF18A and added novel insight on the role of estrogen in regulation of MS-KIF18A expression both in vivo and in vitro

Estrogen effects on MS-KIF18A protein expression and turnover.

<p>(A) Lysates of MBA-15 cells challenged with 10⁻⁸ M 17βE₂ for 1 h, 2 h, 4 h and 20 h were analyzed by WB with anti-MS-KIF18A and compared to the untreated control at 1 h (C 1 h) and 20 h (C 20 h). (B) MBA-15 cells were pretreated with 10⁻⁸ M 17βE₂ for duration of 6 h, 24 h or 48 h, metabolic labeled with Met/Cis-S³⁵, chased for various time periods from 1 h to 36 h, lysed, IPed with anti-MS-KIF18A and loaded on SDS-PAGE gel. (C) A 100 kDa protein confirmed as MS-KIF18A by IP and WB with anti-MS-KIF18A. Results are of representative experiment of a series repeated three times.</p

Interaction between NF-κB and MS-KIF18A or ERα.

<p>IP with anti-p50 (1) with anti-p65 (2) WB performed with anti-MS-KIF18A (A) with anti-ERα (B). Results revealed an association between MS-KIF18A and p50, but not with p65 while ERα interacts with both forms of NF-κB; p65 and p50. Results demonstrate a representative experiment of three independent repeats.</p

Primers used for mRNA expression analyzed by PCR.

<p>Primers used for mRNA expression analyzed by PCR.</p

Estrogen-dependent association between MS-KIF18A and ERα.

<p>MBA-15 cell lysates were IPed with anti-ERα or anti-MS-KIF18A, and analyzed by WB. Results are of representative experiment of a series repeated five times.</p

Sub-cellular distribution of ERα and MS-KIF18A in cells fractionated to cytoplasm (C) and nuclear/membrane (N/M) compartments.

<p>Whole lysates (A) and IP (B) of fractionated cells were analyzed with anti-MS-KIF18A and anti-ERα. Results demonstrate a representative experiment of four independent repeats.</p

Association between MS-KIF18A and ERα recombinant proteins.

<p>Schematic illustration of full length MS-KIF18A (A); truncated MS-KIF18A construct 1–635 aa (B); truncated MS-KIF18A construct 635–898 aa (C). (D–F) Co-IP experiments of MS-KIF18A constucts with recombinant ERα and WB with monoclonal anti-MS-KIF18A (1) and anti-ERα (2). Full length MS-KIF18A (D), MS-KIF18A constucts 1–635 aa (E), MS-KIF18A constucts 635–898 (F). Pull down with beads only (1); IP with anti-MS-KIF18A and WB with anti-ERα (2) (G). The results are representative from the set of at least three independent experiences.</p

Luciferase measurements of MS-KIF18A promoter activity.

<p>(A) Schematic illustration of MS-KIF18A promoter-luciferase reporter constructs. MCF-7 cells transfection with MS-KIF18A promoter cloned in luciferase reporter plasmid (pGLuc-K) or promoter less pGL3-basic along with β-galactosidase vector. (B) Cells treated (black bars) or not (white bars) with 17βE₂ (10⁻⁸ M) for 24 h; (C) 17βE₂ (10⁻⁸ M) or/and ICI-182,780 (10⁻⁷ M) were added to the cultures for 1 h (white bars) or 24 h (gray bars). Promoter activities are expressed as luciferase values normalized for β-galactosidase levels. A value of 100% was given to the basal promoter activity elicited by the pGLuc-K construct in the absence of any treatment. Results are mean±SD of 3 independent experiments, performed in duplicates.</p

Chip assay of ERα and AP-1 binding to MS-KIF18A promoter.

<p>A-C Bar histogram of qPCR analysis of amplified MS-KIF18A promoter in MCF-7 ChIPed by anti-ERα (A) or by anti-pcJun (B), and in MBA-15 cells were ChIPed by anti-ERα (C). All the results presented as mean values +/− SD obtained from three different experiments each performed in triplicates for each data point.</p

MS-KIF18A mRNA expression <i>in vivo</i>.

<p>Total RNA from bone marrow cells were harvested from mice (A) and rats (B, C) and analyzed by qRT-PCR. (A) mRNA expression in bone marrow cells derived from young 4 month (white bars) and old 12 month (black bars) male mice; (B) Young 3 month (white bars) and 14 month old (black bars) male and OVX female rats; (C) Sham, OVX and OVX+E₂ female rats. MS-KIF18A mRNA expression was normalized to G3PDH expression levels. Results are presented as mean values +/− SD obtained from triplicates for each data point.</p