Biological and Pharmaceutical Bulletin
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Characterization of the Role of MANF in Regulating the Secretion of CRELD2
Kentaro Oh-hashiJunpei NorisadaYoko HirataKazutoshi Kiuchi
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2015 Volume 38 Issue 5 Pages 722-731

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Abstract

We recently demonstrated that the secretion of two novel endoplasmic reticulum (ER) stress-inducible proteins, cysteine-rich with epidermal growth factor (EGF)-like domains 2 (CRELD2) and mesencephalic astrocyte-derived neurotrophic factor (MANF), are oppositely regulated by the overexpression of 78 kDa glucose-regulated protein (GRP78). In the present study, we found that the co-transfection of CRELD2 and MANF remarkably enhanced the secretion of CRELD2 without affecting the expression level of GRP78. To identify the structural features of CRELD2 and MANF involved in this process, we generated several CRELD2 and MANF expression constructs. The deletion of the four C-terminal amino acids, either REDL in CRELD2 or RTDL in MANF, abolished the increased secretion of CRELD2 induced by the co-expression of MANF. The deleted mutation of MANF partially abolished the increased secretion of wild type CRELD2 (wtCRELD2) as a positive action of wild type MANF (wtMANF), even when we added the amino acid sequence RTDL at the C-terminus of each mutated MANF construct. Enhanced green fluorescent protein (EGFP), which was tagged with the signal peptide sequence at the N-terminus and four C-terminal amino acids (KEDL, REDL or RTDL), were retained intracellularly, but they did not enhance the secretion of wtCRELD2. Taken together, our data demonstrate that MANF is a factor in regulating the secretion of CRELD2 through four C-terminal amino acids, RTDL and REDL, and the fluctuation of intracellular MANF seems to potentiate the secretion of CRELD2.

The folding and modification of newly synthesized transmembrane and secretory proteins in the endoplasmic reticulum (ER) is maintained by a variety of mechanisms.1,2) Under some pathophysiological conditions, certain ER functions become disordered and then unfolded and/or misfolded proteins are accumulated in the ER.3,4) Abnormal protein retention results in ER stress and activation of the three canonical ER-resident stress sensors; protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK),5) inositol-requiring enzyme 1 (IRE1)6) and activating transcription factor 6 (ATF6),7) induces a variety of genes.812) Among them, growth arrest and DNA damage-inducible protein 153 (GADD153) is well-known to promote stress-induced cell death by activating caspases.8,9) ER resident molecular chaperones such as 78 kDa glucose-regulated protein (GRP78) are reported to alleviate the stress by properly folding and degrading unfolded proteins and attenuating ER stress signals.10,11)

We previously identified the cysteine-rich with epidermal growth factor (EGF)-like domains 2 (CRELD2) gene as a novel ER stress-inducible gene and demonstrate that ATF6 positively regulates the transcription of the CRELD2 gene through a well-conserved ER stress response element (ERSE) in the proximal region of the mouse CRELD2 promoter.12) Furthermore, we have characterized the intracellular traffic and secretion of CRELD213,14); however, the precise functions of the CRELD2 protein remain to be determined.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) was first identified as arginine-rich, mutated in early stage of tumors (Armet) with a high mutation rate in various tumors.15,16) After that, Petrova et al. reported a neurotrophic action of Armet for dopaminergic neurons17) and Armet is referred to as MANF, even though the precise mechanisms underlying this neuroprotective effect are not well understood.1721) On the other hand, it is reported that MANF is expressed in several types of cells and tissues, and is induced by the unfolded protein response via the ER stress response element-II (ERSE-II).2224) In addition to the transcriptional regulation of CRELD2 and MANF, we have been characterizing the post-transcriptional regulation of them and found their secretion is oppositely regulated by GRP78 overexpression.13,25)

In the present study, we found that MANF is a key factor regulating the secretion of CRELD2, which was affected by transiently overexpression of MANF. Furthermore, we characterized the structural features of CRELD2 and MANF by generating several CRELD2 and MANF replacement and deletion constructs to understand the mechanism of MANF-induced CRELD2 secretion. We demonstrate that the overexpression of intracellular MANF influences protein transport and secretion, especially that of CRELD2. Our results further indicate that the relationships between CRELD2 and MANF under normal and pathophysiological conditions may affect the onset and progression of several ER stress-related diseases.

MATERIALS AND METHODS

Materials

The primary antibodies used were as follows: goat polyclonal anti-MANF which was raised against the human MANF (22–179 aa) (R&D Systems, U.S.A.), rabbit polyclonal anti-MANF which was raised against the 12 aa near N-terminal of the human MANF (Abcam, U.K.), goat polyclonal anti-CRELD2 which was raised against the mouse CRELD2 (23–350 aa) (R&D Systems, U.S.A.), mouse monoclonal anti-KDEL proteins including GRP78 and 94 kDa glucose-regulated protein (GRP94) (Medical & Biological Laboratories, Japan), mouse monoclonal anti-Myc-epitope (9E10) (Santa Cruz Biotechnology, U.S.A.), mouse monoclonal anti-Flag-epitope (Sigma-Aldrich, U.S.A.) and mouse monoclonal anti-green fluorescent protein (GFP) (Roche Applied Science, Germany) and mouse monoclonal anti-actin (Calbiochem, U.S.A.).

Plasmid Construction

Mouse wtCRELD2 (NM_029720.2) and wtMANF (NM_029103.3) constructs were prepared by polymerase chain reaction (PCR) as previously described.1214,25) Each variant of the mouse CRELD2 gene was amplified by PCR and then cloned into the pcDNA3.1 vector to generate the following constructs: ΔCCRELD2 (CRELD2 lacking the four C-terminal amino acids REDL). CRELD2 variants with the C-terminal REDL replaced with KDEL (CRELD2KDEL) or RTDL (CRELD2RTDL) were also prepared by PCR and inserted into the pcDNA3.1 vector. Flag (F), EGFP-Flag (EFGP) or Myc epitopes were inserted downstream of the putative signal sequence (23 amino acids) of full-length (FL) CRELD2 [SP-F-FLCRELD2, SP-EGFP-FLCRELD2] or upstream of the four C-terminal amino acids [FLCRELD2-Myc-REDL], respectively. Full-length (FLCRELD2-MH) was inserted into pcDNA3.1 Myc/His vector. Each mouse MANF construct was amplified by PCR and then cloned into the pcDNA3.1 vector: MANF lacking the four C-terminal amino acids (RTDL) (ΔCMANF) and MANF with the C-terminal RTDL replaced with KDEL (MANFKDEL) or REDL (MANFREDL). The Myc epitope was inserted upstream of the four C-terminal amino acids [FLMANF-Myc-RTDL] to produce Δ1MANF-Myc-RTDL (1–26/75–179 aa) and Δ2MANF-Myc-REDL (1–79/122–179 aa) constructs. The Flag epitope was inserted downstream of the putative signal sequence (22 amino acids) of full-length and modified MANF [SP-F-FLMANF or SP-F-Δ3MANF-RTDL (1–123 aa+RTDL)] or added to MANF lacking the putative signal sequence (F-ΔNMANF, 23–179 aa). Each enhanced green fluorescent protein (EGFP) construct with or without the putative signal sequence of mouse CRELD2 (1–23 aa) and the 3 variants of the four C-terminal amino acids, KDEL, REDL and RTDL, was amplified by PCR and inserted into the pcDNA3.1 vector. Mouse GRP78 (NM_001163434.1) construct was prepared by PCR as previously described.13)

Cell Culture and Treatment

HEK293 and COS7 cells were maintained in Dulbecco’s modified Eagle’s minimum essential medium (DMEM) containing 8% fetal bovine serum. Transfections of each expression vector used in this study were performed using the Lipofectamine-Plus reagent (Life Technologies, U.S.A.) according to the manufacturer’s instructions as described previously.12,13,25) For transient overexpression of each gene, same number of cells in 12-well plate or glass-bottomed dishes were transfected with the indicated constructs and cultured for 24 h. The culture medium was then replaced with an equal amount of fresh, serum-free DMEM and incubated for an additional 8 or 12 h.

Western Blot Analysis

Cells from each well were lysed with homogenate buffer [20 mM Tris–HCl (pH 8.0) containing 137 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/mL leupeptin and 10 µg/mL pepstatin A] as described previously.12,13,25) After determining the protein concentrations by a Bradford Reagent (BioRad, U.S.A.), cell lysates were dissolved in sodium dodecyl sulfate (SDS)-Laemmli sample buffer [62.5 mM Tris–HCl (pH 6.8), 2% SDS and 10% glycerol], and equal amounts of cell lysates in each experiment were prepared. To detect MANF, CRELD2 and EGFP in the culture medium, equal amounts of each culture medium was resuspended in SDS-Laemmli sample buffer. In each experiments, equal amounts of each sample from lysates and culture medium were separated on 8.0 or 15.0% SDS-polyacrylamide electrophoresis gels, immunoblotted onto polyvinylidene difluoride membranes (GE Healthcare Bioscience, U.S.A.) and identified by enhanced chemiluminescence using antibodies against CRELD2, MANF, KDEL proteins, the Myc epitope, the Flag epitope, GFP or actin. More than three independent cultures were performed to confirm reproducibility and amounts of the secreted CRELD2 were analyzed by NIH image.

Fluorescence Microscopy

Twenty-four hours after SP-EGFP-FLCRELD2, wtCRELD2, SP-F-FLMANF or wtMANF was transfected into HEK293 or COS7 cells on glass-bottomed dishes or glass coverslips, the culture medium was replaced with serum-free DMEM, and the cells were incubated for an additional 8 or 12 h. The living HEK293 cells expressing SP-EGFP-FLCRELD2 were observed by fluorescence microscopy (OLYMPUS, Japan). COS7 cells expressing the indicated CRELD2 and MANF on glass coverslips were fixed with PBS containing 4% paraformaldehyde for 10 min as described previously.12,13) For the detection of wtCRELD2 and Flag-tagged MANF, fixed cells were permeabilized with PBS containing 0.2% Triton X-100 for 3 min. The coverslips were then incubated with anti-CRELD2 polyclonal and anti-Flag (M2) monoclonal antibodies overnight at 4°C, respectively. After washing, the coverslips were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-goat immunoglobulin G (IgG) (Vector Labs, U.K.) and Texas Red-conjugated anti-mouse IgG (Vector Labs, U.K.) as secondary antibodies. Cell nuclei were stained with Hoechst 33823 and the cells were mounted in Vectashield (Vector Labs, U.K.) and observed by fluorescence microscopy (KEYENCE, Japan).

Statistical Analysis

Statistical analyses were carried out using one-way ANOVA followed by Fisher PLSD method. A p<0.05 was considered to be statistically significant.

RESULTS

We previously detected three novel ER stress-inducible genes, CRELD2,12,13) MANF24,25) and cation transport regulator homolog 1 (Chac1),26) in thapsigargin-treated Neuro2a cells using microarray analysis and characterized their transcriptional regulation and protein features. Among them, CRELD2 is a secretory glycoprotein, and its secretion is positively regulated by GRP78.12,13) Conversely, MANF secretion is down-regulated by GRP78 overexpression.25) CRELD2 and MANF proteins have a series of four C-terminal amino acids that are well-conserved among several species, [R/H]EDL in CRELD2 and R[T/S][D/E]L in MANF, and these amino acids resemble a well-known ER resident signal, KDEL. Deletion of the four C-terminal amino acids and/or addition of a tag peptide after the C-terminus markedly influences their intracellular retention and secretion.13,25) These results indicate that the four C-terminal amino acids of CRELD2 and MANF, which are similar to those in several ER resident proteins, play an important role in retaining each protein within the ER lumen. Then, we tried to characterize the relationship between these two proteins using HEK293 and COS7 cells. Both cell-lines expressed intrinsic CRELD2 and MANF mRNA (Supplementary Fig. 1), but the expression levels of these two proteins were below detection limits in our experimental conditions. Therefore, we co-transfected both of the genes into these cell-lines to characterize the molecular features of these two secretory factors clearly in this study.

First, using various CRELD2 expression vectors, we determined which molecular structures in CRELD2 are responsible for its secretion induced by wild type MANF (wtMANF) co-transfection (Fig. 1A). As shown in Fig. 1B, the co-transfection of wild type CRELD2 (wtCRELD2) with wtMANF caused the increased secretion of wtCRELD2 from the co-transfected cells compared to cells transfected with wtCRELD2 alone. The increased secretion of wtCRELD2 upon wtMANF overexpression was almost comparable to the increased secretion observed upon GRP78 overexpression, but the amounts of GRP78 and GRP94 in cell lysates were hardly affected by MANF and/or CRELD2 overexpression. Then, we examined the changes in CRELD2 secretion upon overexpression of modified MANF in more detail.

Fig. 1. The Overexpression of wtMANF Induced CRELD2 Secretion from HEK293 Cells

A) Schematic of the mouse CRELD2 expression constructs used in this study. SP indicates a signal sequence at the N-terminus of protein translated in the ER. Open and hatched boxes represent an EGF-like domain and furin-like repeats, respectively. B) Twenty-four hours after transfection of the wild type CRELD2 (wtCRELD2), wild type MANF (wtMANF), GRP78 constructs or an empty vector, the culture medium was replaced with serum-free DMEM, and the cells were incubated for an additional 12 h. The amount of the indicated proteins in each lysate or in the culture medium was determined by Western blotting as described in Materials and Methods. Representative results are shown in this figure and comparable results were achieved in independent experiments. Blots were quantified with a NIH-imaging. Numbers below the lanes indicate the relative amount of secreted wtCRELD2 and wtMANF as compared to those of wtCRELD2 or wtMANF alone transfected cells.

As shown in Fig. 2A, CRELD2 protein lacking the original four C-terminal amino acids REDL (ΔCCRELD2) was highly secreted into the extracellular space, but the secretion ratio was not affected by wtMANF co-transfection at all. Replacing CRELD2 with the canonical ER-resident signal KDEL almost abolished its secretion, even when wtMANF was overexpressed. CRELD2RTDL, containing the MANF-type C-terminal peptide (RTDL), was secreted significantly higher than wtCRELD2 in the absence of wtMANF, and the overexpression of wtMANF also enhanced the secretion of CRELD2RTDL. In addition, we prepared CRELD2 constructs having Flag- or Myc/His-epitope tags at the N- or C-terminus and evaluated the secretion ratio of each protein with or without wtMANF overexpression (Fig. 2B). CRELD2 protein with a Flag-epitope just downstream of the signal sequence (SP-F-FLCRELD2) showed almost a similar amount of secretion as wtCRELD2, but CRELD2 protein with Myc/His-epitope tags at the C-terminus (FLCRELD2-MH) resembled the C-terminal deletion mutant of CRELD2 (ΔCCRELD2) in its secretory profile (Fig. 2A). FLCRELD2-Myc-REDL with a Myc-epitope upstream of the REDL sequence showed a significant increase in spontaneous secretion compared with wtCRELD2 without wtMANF, and also responded to the overexpression of wtMANF significantly.

Fig. 2. Effect of wtMANF on the Mutant CRELD2 Secretion from HEK293 Cells

AB-a) Schematic of the mouse CRELD2 expression constructs. AB-b) The indicated constructs were transfected into HEK293 cells and incubated as described in Fig. 1. The expression of the indicated proteins in each lysate or in the culture medium was determined by Western blotting as described in Materials and Methods. Representative results are shown in this figure. The dotted lines indicate the boundary between the two distant lanes in the same gel image. AB-c) Blots of the CRELD2 in the culture medium were quantified with a NIH-imaging. The values indicate the relative amount of secreted CRELD2 as compared to the amount of the secreted wtCRELD2 from the wtMANF-cotransfected cells. The values represent the mean±S.E.M. from 3 independent cultures. The data were analyzed by a one-way ANOVA followed by the Fischer PLSD test to evaluate the effects of wtMANF overexpression on the indicated CRELD2 secretion. The values marked with an asterisk are significantly different from the value of the secreted CRELD2 in the wtMANF-transfected cells (p<0.05).

Next, using various MANF expression constructs (Fig. 3A), we investigated which molecular structures in MANF were responsible for the secretion of wtCRELD2. As shown in Fig. 3B, MANFKDEL and MANFREDL having the different C-terminal end instead of RTDL also significantly increased wtCRELD2 secretion. In contrast, deletion of the putative N-terminal signal peptide (F-ΔNMANF) or ER resident signal (ΔCMANF) from full-length MANF did not show any significant effect on the wtCRELD2 secretion when co-expressed. However, SP-F-FLMANF or FLMANF-Myc-RTDL was able to elevate wtCRELD2 secretion. We further generated three modified MANF constructs lacking a portion of the N- or C-terminal regions (Δ1MANF-Myc-RTDL, Δ2 MANF-Myc-RTDL and SP-F-Δ3MANF-RTDL) to examine their effects on wtCRELD2 secretion (Fig. 4). The wtCRELD2 secretion by co-transfection of each deletion mutant of MANF was significantly lower than that by SP-F-FLMANF or FLMANF-Myc-RTDL overexpression, but each of the mutant slightly increased the wtCRELD2 secretion compared to that with an empty vector (mock). In this condition, neither of MANF mutants was secreted into the extracellular space despite each mutant protein being expressed, as each was clearly detected by Western blot analysis of cell lysates.

Fig. 3. Effects of N- or C-Terminal Mutated MANF Overexpression on wtCRELD2 Secretion from HEK293 Cells

A) Schematic of the mouse MANF expression constructs. B) The indicated constructs were transfected into HEK293 cells and incubated as described in Fig. 1. The expression of the indicated proteins in each lysate or in the culture medium was determined as described in Materials and Methods. Representative results are shown in this figure. C) Blots of the CRELD2 in the culture medium were quantified and analyzed as described in Fig. 2. The values represent the mean±S.E.M. from 3 independent cultures. The values marked with an asterisk are significantly different from the value of the secreted wtCRELD2 in the mock-transfected cells (p<0.05).

Fig. 4. Effects of Truncated Mutant MANF Overexpression on wtCRELD2 Secretion from HEK293 Cells

AB-a) Schematic of the mouse MANF expression constructs. b) The indicated constructs were transfected into HEK293 cells and incubated as described in Fig. 1. The expression of the indicated proteins in each lysate or in the culture medium was determined as described in Materials and Methods. Representative results are shown in this figure. The dotted lines indicate the boundary between the two distant lanes in the same gel image. c) Blots of the CRELD2 in the culture medium were quantified and analyzed as described in Fig. 2. The values represent the mean±S.E.M. from 3 independent cultures. The values indicate the relative amount of secreted wtCRELD2 as compared to those of the secreted wtCRELD2 from the FLMANF-Myc-RTDL (A) or SP-F-FLMANF (B)-cotransfected cells. The values marked with an asterisk are significantly different from the value of the secreted wtCRELD2 in the FLMANF-Myc-RTDL (A) or SP-F-FLMANF (B)-transfected cells, respectively (p<0.05).

We next observed the intracellular localization of SP-EGFP-FLCRELD2, in which EGFP is located downstream of the putative signal sequence. As shown in Figs. 5A and B, overexpression of wtMANF increased SP-EGFP-FLCRELD2 secretion; however, no change in SP-EGFP-FLCRELD2 intracellular localization was observed by fluorescent microscopy. We further transfected wtCRELD2 with or without SP-F-FLMANF and wtMANF into COS7 cells. As shown in the above experiments using HEK293 cells, the secretion of wtCRELD2 from COS7 cells was also up-regulated by the SP-F-FLMANF overexpression (Fig. 5C). Similarly, the co-transfection of wtMANF into COS7 cells also increased the wtCRELD2 secretion as observed in HEK293 cells. We further observed the partial co-localization of wtCRELD2 and SP-F-FLMANF within COS7 cells by immunostaining of these two proteins (Fig. 5D).

Fig. 5. Effect of MANF Overexpression on the Intracellular Localization and Secretion of CRELD2

After the transfection of EGFP-tagged CRELD2 (SP-EGFP-FLCRELD2) into HEK293 cells (A and B) or wtCRELD2 into COS7 cells (C and D) with or without the indicated MANF genes, the culture medium was replaced with serum-free DMEM. The cells were incubated for 12 h (A) or 8 h (D) and fluorescent images were acquired as described in Materials and Methods. B, C-a) The expression of the indicated proteins in each lysate or in the culture medium was determined as described in the Materials and Methods. B) Numbers below the lanes indicate the relative amount of secreted SP-EGFP-FLCRELD2 as compared to the amount of the secreted SP-EGFP-FLCRELD2 alone-transfected cells. C-b) The values indicate the relative amount of secreted wtCRELD2 as compared to the amount of the secreted wtCRELD2 from the wtMANF-cotransfected cells. The values represent the mean±S.E.M. from 3 independent cultures. The data were analyzed as described in Fig. 2. The values marked with an asterisk are significantly different from the value of the secreted wtCRELD2 in the wtMANF-transfected cells (p<0.05).

Finally, we investigated the effects of other secretory proteins on the MANF-induced CRELD2 secretion using EGFP with the signal sequence of CRELD2 at the N-terminus (SP-EGFP) and the ER-resident signals at the C-terminus. The secretion of SP-EGFP without the four amino acids at the C-terminus was not elevated by overexpression of wtMANF (Figs. 6A, B). Furthermore, we analyzed the secretion of wtCRELD2 when co-transfected with SP-EGFPs containing different C-terminal amino acids (SP-EGFPKDEL, SP-EGFPREDL or SP-EGFPRTDL) (Figs. 6A, C). In contrast to SP-EGFP, SP-EGFPREDL, SP-EGFPRTDL and SP-EGFPKDEL were hardly detected in the culture medium, and the cells expressing each of these SP-EGFP variants showed stronger fluorescence intensity than those expressing SP-EGFP without the four C-terminal amino acids (Supplementary Fig. 2). We found that the secretion of wtCRELD2 was not elevated by the co-transfection of any SP-EGFP construct.

Fig. 6. The Overexpression of ER-Localized EGFP Did Not Induce wtCRELD2 Secretion

A) Schematic of the different EGFP constructs used in this study. SP indicates the signal sequence of mouse CRELD2 at the N-terminus. B and C) Twenty-four hours after transfection of the indicated genes, the culture medium was replaced with serum-free DMEM, and the cells were incubated for an additional 12 h. The amounts of the indicated protein in the lysate or culture medium were determined as described in Materials and Methods.

DISCUSSION

MANF has been reported to prevent neuronal cell death caused by several neurotoxic stimuli, but the precise mechanisms are still unclear. On the other hand, the MANF gene is also reported as an ER stress inducible factor regulated by ATF6 and/or X-box binding protein 1 (XBP1).2224) Because ATF6 and/or XBP1 regulate several genes which participate in protein folding, transport and degradation within the ER,11,27) we have been investigating whether MANF participates in some of these processes inside cells. Interestingly, we first found that the MANF overexpression positively regulated the secretion of CRELD2, which we recently identified as a novel ER stress-inducible factor.12) It was previously reported that GRP78 overexpression modulates the expression and secretion of CRELD2 and MANF.13,25) However, the transient overexpression of MANF and/or CRELD2 hardly changed the expression of GRP78 and GRP94 protein in HEK293 cells. These results suggest that the increase in the CRELD2 secretion by MANF overexpression might not be simply due to the expression of intrinsic ER resident chaperones such as GRP78 and GRP94, which are known as ER stress-inducible factors. On the other hand, it is reported that the secretion of CRELD2 and MANF is not accompanied with the magnitude of ER stress.12,13,25,28,29) It is therefore considered that there would be more complicated relationships between the CRELD2 and MANF proteins to regulate their secretion.

Based on our previous reports that the well-conserved four C-terminal amino acids in both MANF and CRELD2 are critical for their intracellular retention and secretion,12,13,25) our mutational analysis revealed that the increased secretion of CRELD2 by MANF overexpression also depends on the C-terminal amino acids of both proteins. It is demonstrated that many ER resident proteins is retained within the ER by retrieval transport from the Golgi via KDEL receptors, which specifically recognize the four C-terminal amino acids in ER resident proteins.30,31) However, the increased secretion of wtCRELD2 upon wtMANF overexpression is not simply a result of competition between the two proteins for KDEL receptors during ER-Golgi transport because wtMANF overexpression increased the secretion of wtCRELD2 but not SP-EGFPs. In addition, we observed that the intracellular amount of SP-F-FLMANF is lower than that of wtMANF in both cell-lines, but both MANF induced the CRELD2 secretion to a similar extent (Figs. 3, 5C). Considering the different amount of each MANF protein within cells in the current study, it is thought that their intracellular amounts were sufficient to induce the CRELD2 secretion. The four C-terminal amino acids of MANF rather determined this unique feature of MANF. Therefore, not only structural but also quantitative analysis of MANF and CRELD2 would uncover the precise relationships between two.

We also examined the intracellular localization of CRELD2 with or without wtMANF. The secretory wtCRELD2 and CRELD2 having the C-terminal modifications were stained throughout cells and the staining in the perinuclear region was relatively intensive (Supplementary Fig. 3). However, no apparent change in the localization of SP-EGFP-FLCRELD2 and wtCRELD2 was observed by MANF overexpression. Considering their secretory features and colocalization within cells, we tried to evaluate the direct interaction between CRELD2 and MANF by immunoprecipitation, but it was not detected in our experimental conditions (data not shown). Therefore, further studies about the relationship between CRELD2 and MANF proteins are required to uncover their roles in regulating several intracellular events, including retention, transport and secretion of proteins in the organelle.

Recently, MANF has been reported to have a trophic effect on peripheral non-neuronal cells, such as cardiomyocytes and several cell lines.23,32) In contrast, Palgi et al. reported that various genes, including some genes related to protein processing in the ER, fluctuated in three types of MANF Drosophila mutants.33) On the other hand, CRELD2 has been reported to mediate the intracellular trafficking of acetylcholine receptor α4 and β2 subunits.34) Therefore, both CRELD2 and MANF may be in the same cargo to regulate the transport of certain secretory factors and transmembrane proteins from the ER to the Golgi apparatus. In the present study, we first demonstrated that the function of MANF in regulating CRELD2 secretion is dependent on the C-terminal KDEL-like motif by the transient overexpression of CRELD2 and MANF. However, our study clearly demonstrate that the effect of MANF on CRELD2 secretion might not be simply due to competition between each C-terminal motif for binding to KDEL receptors. Henderson et al. have reported KDEL receptors regulate the transport and secretion of MANF,35) but the factors regulating the relationship between CRELD2 and MANF secretion remain to be determined. Therefore, further characterization of their relationship including KDEL receptors is required to reveal the novel mechanisms underlying the regulation of protein transport and secretion within the ER and Golgi apparatus. Very recently, it was demonstrated that CRELD2 induces the osteogenic differentiation of mesenchymal stem cells.36) This suggests that CRELD2 acts as a humoral factor along with MANF, though the precise signaling pathways have not been fully identified. In the last few years, it has been reported that the expression of CRELD2 and MANF are elevated in several physiological conditions, which are not restricted to the central nervous system.23,3739) Therefore, further characterization of MANF and CRELD2 inside and outside cells under pathophysiological conditions may give new insights into the onset and progression of ER stress-related diseases.

Acknowledgments

This work is in part supported by the Research Foundation for the Electrotechnology of Chubu and the Suzuken Memorial Foundation.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

REFERENCES
 
© 2015 The Pharmaceutical Society of Japan
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