High-level secretion of native recombinant human calreticulin in yeast

Additional file 2. Elution profiles of recombinant CRT in anion-exchange chromatography. Separation of CRT from S. cerevisiae (A and B) was performed on a pre-packed 5 ml Q-Sepharose column (GE LifeSciences cat. no. 17-5156-01), equilibrated with 20 mM l-His, pH 5.5, 100 mM NaCl buffer, in 15 column volumes linear gradient from 100 mM to 500 mM NaCl. Separation of CRT from P. pastoris (C and D) was performed on a 15 ml Q-Sepharose sorbent (GE LifeSciences cat. no. 17-0510-10) packed into the XK 16/20 column (GE Lifesciences, cat. no. 28-9889-37), equilibrated with 20 mM l-His, pH 5.5, 100 mM NaCl buffer, in 10 column volumes linear gradient from 100 to 500 mM NaCl. Elution profiles (A and C) and SDS-PAGE gels of corresponding fractions (B and D) are shown. Dashed lines define fractions that were pooled and used for further experiments with purified protein

Estrogen and progesterone regulate p27kip1 levels via the ubiquitin-proteasome system: pathogenic and therapeutic implications for endometrial cancer.

The levels of proteins that control the cell cycle are regulated by ubiquitin-mediated degradation via the ubiquitin-proteasome system (UPS) by substrate-specific E3 ubiquitin ligases. The cyclin-dependent kinase inhibitor, p27kip1 (p27), that blocks the cell cycle in G1, is ubiquitylated by the E3 ligase SCF-Skp2/Cks1 for degradation by the UPS. In turn, Skp2 and Cks1 are ubiquitylated by the E3 ligase complex APC/Cdh1 for destruction thereby maintaining abundant levels of nuclear p27. We previously showed that perpetual proteasomal degradation of p27 is an early event in Type I endometrial carcinogenesis (ECA), an estrogen (E2)-induced cancer. The present studies demonstrate that E2 stimulates growth of ECA cell lines and normal primary endometrial epithelial cells (EECs) and induces MAPK-ERK1/2-dependent phosphorylation of p27 on Thr187, a prerequisite for p27 ubiquitylation by nuclear SCF-Skp2/Cks1 and subsequent degradation. In addition, E2 decreases the E3 ligase [APC]Cdh1 leaving Skp2 and Cks1 intact to cause p27 degradation. Furthermore, knocking-down Skp2 prevents E2-induced p27 degradation and growth stimulation suggesting that the pathogenesis of E2-induced ECA is dependent on Skp2-mediated degradation of p27. Conversely, progesterone (Pg) as an inhibitor of endometrial proliferation increases nuclear p27 and Cdh1 in primary EECs and ECA cells. Pg, also increases Cdh1 binding to APC to form the active E3ligase. Knocking-down Cdh1 obviates Pg-induced stabilization of p27 and growth inhibition. Notably, neither E2 nor Pg affected transcription of Cdh1, Skp2, Cks1 nor p27. These studies provide new insights into hormone regulation of cell proliferation through the UPS. The data implicates that preventing nuclear p27 degradation by blocking Skp2/Cks1-mediated degradation of p27 or increasing Cdh1 to mediate degradation of Skp2-Cks1 are potential strategies for the prevention and treatment of ECA

Identical responses to E2 and Pg by ECA cell lines and endometrial epithelial cells (EECs).

<p>A, E2; B, Pg treatment of primary EECs: normal EECs from proliferative phase were treated with either E2 without or with ICI or treated with Pg/E2 with or without RU486, cell lysates prepared and immunoblotted for protein levels shown as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046072#s4" target="_blank">Materials and Methods</a>. A and B are different patients showing different basal levels of p27 and Cdh1. C, D: E2, E2 plus Pg treatment, cell proliferation (by MTS assay): parallel experiment for patient #2, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046072#s4" target="_blank">Materials and Methods</a>; *p<0.05. E, E2, Pg treatment, subcellular fractionation: Primary EECs from proliferative phase were treated with E2 or Pg/E2, subjected to subcellular fractionation, and immunoblot analysis for the protein levels shown and performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046072#s4" target="_blank">Materials and Methods</a>. F, Pg, Primary ECA cells (grade III): Cells were isolated, treated with Pg/E2 in the presence or absence of RU486, subjected to subcellular fractionation followed by immunoblot analysis for Cdh1 and p27, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046072#s4" target="_blank">Materials and Methods</a>.</p

Estrogen (E2) and progesterone (Pg) inversely regulate the levels of nuclear p27 and Cdh1, Skp2, and Cks1 proteins of the ubiquitin-proteasome system for cell cycle control.

<p>Cdc14 phosphatase keeps Cdh1 bound to APC; this is the E3 ligase that ubiquitylates Skp2/Cks1 of the SCF complex. E2 decreases nuclear [APC]Cdh1 and induces phosphorylation of p27 on T187 signaling its ubiquitylation by [SCF]-Skp2/Cks1. The decrease in [APC]Cdh1 prevents ubiquitin-mediated degradation of Skp2/Cks1 allowing [SCF]Skp2/Cks1 to ubiquitylate p27 causing its proteasomal degradation; cells progress through S phase. Pg increases [APC]Cdh1, which ubiquitylates [SCF]Skp2/Cks1 causing their degradation blocking p27 degradation; cells are blocked in G1. Ovals are E3 ligases; green small squares are chains of ubiquitin (Ub); small yellow circles indicate phosphorylation (P). Center Table: Slight (<15%); v* = variable, slightly up in ECC-1 and variable EECs. Cdh1 was not present in the cytoplasm in ECC-1 cells or EECs but was found in primary ECA cells (n = 2/3).</p

E2 induces ERK-dependent phosphorylation of p27 at Thr187.

<p>A, E2 dose-response: EEC-1 were treated with E2 and immunoblot analysis performed using rabbit anti-human phospho-p27 (p-p27[T187]), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046072#s4" target="_blank">Materials and Methods</a>. B, E2 time course: ECC-1 cells were treated with E2 and protein levels determined by immunoblotting as in A. C, E2, phospho-ERK: ECC-1 cells were treated with E2 in the presence of U0126 or ICI and cell lysates immunoblotted for p-p27(T187), phospho ERK (p-ERK), and total ERK, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046072#s4" target="_blank">Materials and Methods</a>. D, E2, Analysis of ERK1 and ERK2 activation following their knock-down with siRNA: ECC-1 cells were transfected with ERK1, ERK2, or control siRNA, treated with E2 in the presence or absence of ICI, followed by cell fractionation, and immunoblotting for total and phospho-ERK-1(p-ERK1) and ERK2 (p-ERK2), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046072#s4" target="_blank">Materials and Methods</a>.</p

Time course: Opposite effect of E2 and Pg on Cdh1 protein levels; Pg and E2 do not affect mRNA levels of Cdh1, Skp2, Cks1 or p27.

<p>A, B, Time course of E2 and Pg [plus E2] treatment of EEC-1 cells. ECC-1 cells were treated with E2 and Pg plus E2, the experiments terminated at the times indicated, lysates prepared, and Cdh1 protein levels determined by immunoblotting, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046072#s4" target="_blank">Materials and Methods</a>. C, D, E2, Pg, mRNA levels of p27, Skp2, Cks1 and Cdh1: ECC-1 cells were treated with E2 or Pg/E2, total RNA extracted at the times shown, and quantitative real-time RT-PCR performed, all in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046072#s4" target="_blank">Materials and Methods</a>. PR and glycodelin primers were used as controls for ER and PR target genes, respectively. Data are presented as an average of two independent experiments.</p

Estrogen (E2) and progesterone (Pg) have opposite effects on cell proliferation and levels of p27, Skp2, and Cks1 proteins.

<p>Details are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046072#s4" target="_blank">Materials and Methods</a> and each Figure. A, E2 dose-response: ECC-1 and KLE cells were treated with E2 and p27, Skp2, and Cks1 protein levels determined by immunoblotting. B, E2 time course: ECC-1 cells were treated with E2 for the times indicated and protein levels determined as above. C, Pg dose-response: ECC-1 cells were treated with Pg/E2 and p27, Skp2 and Cks1 protein levels determined as above. D, Pg time course: ECC-1 cells were treated with Pg/E2 for the times shown and protein levels as above. Densitometric scans of all protein bands are shown by the graphs to the right of each blot. Each band was normalized to actin and then compared with 0 time or untreated control. The data are expressed as relative intensity of each band ± standard deviation. E, E2/Pg cycloheximide (CHX) treatment: ECC-1 cells were treated with E2 for 18 h or with E2 and Pg for 48 h. CHX was added 6 h prior to final harvest. Cells were harvested at 0,1,2, and 6 h time points. CHX alone was the baseline control. Lysates were prepared and immunoblotted for p27 and Skp2 and the levels of each protein band determined by densitometry (band intensity). The percent change within each treatment parameter was calculated based on the zero time point for each group. Protein turn-over was determined by comparison of cells treated with CHX alone compared to CHX in the presence of each hormone at each time point. The line graphs represent the relative intensity of each band normalized to actin for each group. F, G: E2 stimulates and Pg inhibits proliferation: ECC-1 cells were treated with E2 or Pg/E2 and cell proliferation determined by MTS assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046072#s4" target="_blank">Materials and Methods</a>) *p<0.05. H, Cell cycle distribution: ECC-1 cells were treated with E2 or E2/Pg and cell cycle analysis performed as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046072#s4" target="_blank">Materials and Methods</a>.</p

Knock-down of Cdh1 blocks Pg-mediated increase in nuclear p27 and growth inhibition.

<p>A, Pg, Cdh1 knockdown: ECC-1 cells were transiently transfected with Cdh1 siRNA followed by cytoplasmic/nuclear separation, and knockdown efficiency determined in each fraction by comparing control siRNA and Cdh1 siRNA transfected cell lysates. B, Pg, Cdh1 knock-down and subcellular fractionation: transfected cells were treated with Pg/E2 with or without RU486, subjected to cellular fractionation, and immunoblot analysis performed on knock-down and control siRNA cells for proteins shown; all described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046072#s4" target="_blank">Materials and Methods</a>. Densitometric scans (relative intensity) of each protein band representing response levels relative to actin and compared with untreated controls are shown in the graph below. C, Pg, cell proliferation in Cdh1 knockdown cells: cells transfected with Cdh1 or control siRNA were treated Pg/E2 or not treated and proliferation analyzed by MTS assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046072#s4" target="_blank">Materials and Methods</a>; *p<0.05.</p

Subcellular fractionation: opposite Effects of E2 and Pg on Cdh1 protein levels compared to p27, p-p27(T187), Skp2, and Cks1; Pg induces binding of Cdh1 to APC.

<p>A, E2; B, Pg, Cell fractionation and analysis of Cdh1, Skp2, Cks1, p27 proteins and p-p27(T187): Cells were either untreated, treated with E2, E2 plus Lac or treated with Pg/E2 with or without 1 µM Lactacystin (Lac) or RU486. Cells were fractionated and proteins analyzed by immunoblotting, all described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046072#s4" target="_blank">Materials and Methods</a>. The <i>inset in A</i> shows that lactacystin decreases Skp2 protein levels. C., Pg, Cdh1 binding to APC: Cells were treated with Pg/E2, cell lysates immunoprecipitated with anti-Cdh1 followed by immunoblotting with anti-Cdc27, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046072#s4" target="_blank">Materials and Methods</a>. D, diagram depicts APC/Cdh1 as the upstream E3 ligase that ubiquitylates Skp2/Cks1 (of the SCF complex), which is the downstream E3 ligase that ubiquitylates p27 to regulate p27 protein levels.</p