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Intracellular trafficking and secretion of mouse mesencephalic astrocyte-derived neurotrophic factor

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Abstract

Recently, mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to prevent cell death under some pathophysiological conditions. MANF, also referred to as arginine rich, mutated in early stage of tumors (Armet), was identified as an endoplasmic reticulum (ER) stress-inducible factor. Using RT-PCR, we found two variants of MANF mRNA: wild type, which contains exon 1 (wt-MANF), and one lacking exon 1, which is presumably not secreted (ΔΝ-MANF) in Neuro2a cells. The latter has a putative translational start site upstream of the second exon in the mouse MANF gene. Comparing the expression of wt-MANF with that of ΔΝ-MANF, we found that the amount of intracellular ΔΝ-MANF was much lower than that of wt-MANF. Furthermore, ΔΝ-MANF was not detected in the culture medium after its transient transfection into Neuro2a cells. Deletion of several α-helices of mouse MANF decreased its intracellular stability and secretion. Secretion of wt-MANF was almost completely inhibited by either treatment with brefeldin A (BFA), which disrupts the Golgi apparatus structure, or overexpression of a dominant negative Sar1 (Sar1[H79G]), which is reported to impair COPII-mediated transport from the ER to the Golgi apparatus. In addition, the enforced expression of glucose-regulated protein 78 kDa (GRP78) attenuated the secretion of wt-MANF and led to its intracellular accumulation. MANF lacking the four C-terminal amino acids (ΔC-MANF) accumulated at low levels in the cells, but its intracellular level was increased by GRP78 overexpression. The amount of ΔC-MANF in the culture medium was partially down-regulated after co-transfection of GRP78. Substitution of the amino acids RTDL at the C-terminus of mouse MANF with KDEL, the canonical ER localization signal in GRP78, markedly decreased MANF secretion and its secretion was further attenuated by GRP78 overexpression. Taken together, our data show that the secretion of MANF is regulated via COPII-mediated transport and that its C-terminus could be responsible for its retention in the ER through GRP78. The alternate isotype, ΔΝ-MANF, may be less stable in cells than wt-MANF and may not be secreted extracellularly.

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Abbreviations

Armet:

Arginine rich, mutated in early stage of tumor

CRELD2:

Cysteine rich with EGF-like domains 2

ER:

Endoplasmic reticulum

GRP78:

Glucose-regulated protein 78 kDa

MANF:

Mesencephalic astrocyte-derived neurotrophic factor

RT-PCR:

Reverse transcription polymerase chain reaction

References

  1. Gething MJ, Sambrook J (1992) Protein folding in the cell. Nature 355:33–45

    Article  PubMed  CAS  Google Scholar 

  2. Helenius A, Marquardt T, Braakman I (1992) The endoplasmic reticulum as a protein-folding compartment. Trends Cell Biol 2:227–231

    Article  PubMed  CAS  Google Scholar 

  3. Kim I, Xu W, Reed JC (2008) Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov 7:1013–1030

    Article  PubMed  CAS  Google Scholar 

  4. Lindholm D, Wootz H, Korhonen L (2006) ER stress and neurodegenerative diseases. Cell Death Differ 13:385–392

    Article  PubMed  CAS  Google Scholar 

  5. Harding HP, Zhang Y, Ron D (1999) Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397:271–274

    Article  PubMed  CAS  Google Scholar 

  6. Calfon M, Zeng H, Urano F, Till JH, Hubbard SR, Harding HP, Clark SG, Ron D (2002) IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 415:92–96

    Article  PubMed  CAS  Google Scholar 

  7. Zhu C, Johansen FE, Prywes R (1997) Interaction of ATF6 and serum response factor. Mol Cell Biol 17:4957–4966

    PubMed  CAS  Google Scholar 

  8. Rutkowski DT, Kaufman RJ (2003) All roads lead to ATF4. Dev Cell 4:442–444

    Article  PubMed  CAS  Google Scholar 

  9. Oyadomari S, Mori M (2004) Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ 11:381–389

    Article  PubMed  CAS  Google Scholar 

  10. Yamamoto K, Sato T, Matsui T, Sato M, Okada T, Yoshida H, Harada A, Mori K (2007) Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6α and XBP1. Dev Cell 13:365–376

    Article  PubMed  CAS  Google Scholar 

  11. Wang M, Ye R, Barron E, Baumeister P, Mao C, Luo S, Fu Y, Luo B, Dubeau L, Hinton DR, Lee AS (2010) Essential role of the unfolded protein response regulator GRP78/BiP in protection from neuronal apoptosis. Cell Death Differ 17:488–498

    Article  PubMed  CAS  Google Scholar 

  12. Shridhar R, Shridhar V, Rivard S, Siegfried JM, Pietraszkiewicz H, Ensley J, Pauley R, Grignon D, Sakr W, Miller OJ, Smith DI (1996) Mutations in the arginine-rich protein gene, in lung, breast, and prostate cancers, and in squamous cell carcinoma of the head and neck. Cancer Res 56:5576–5578

    PubMed  CAS  Google Scholar 

  13. Evron E, Cairns P, Halachmi N, Ahrendt SA, Reed AL, Sidransky D (1997) Normal polymorphism in the incomplete trinucleotide repeat of the arginine-rich protein gene. Cancer Res 57:2888–2889

    PubMed  CAS  Google Scholar 

  14. Mizobuchi N, Hoseki J, Kubota H, Toyokuni S, Nozaki J, Naitoh M, Koizumi A, Nagata K (2007) ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element. Cell Struct Funct 32:41–50

    Article  PubMed  CAS  Google Scholar 

  15. Petrova P, Raibekas A, Pevsner J, Vigo N, Anafi M, Moore MK, Peaire AE, Shridhar V, Smith DI, Kelly J, Durocher Y, Commissiong JW (2003) MANF: a new mesencephalic, astrocyte-derived neurotrophic factor with selectivity for dopaminergic neurons. J Mol Neurosci 20:173–188

    Article  PubMed  CAS  Google Scholar 

  16. Apostolou A, Shen Y, Liang Y, Luo J, Fang S (2008) Armet, a UPR-upregulated protein, inhibits cell proliferation and ER stress-induced cell death. Exp Cell Res 314:2454–2467

    Article  PubMed  CAS  Google Scholar 

  17. Tadimalla A, Belmont PJ, Thuerauf DJ, Glassy MS, Martindale JJ, Gude N, Sussman MA, Glembotski CC (2008) Mesencephalic astrocyte-derived neurotrophic factor is an ischemia-inducible secreted endoplasmic reticulum stress response protein in the heart. Circ Res 103:1249–1258

    Article  PubMed  CAS  Google Scholar 

  18. Hellman M, Arumäe U, Yu LY, Lindholm P, Peränen J, Saarma M, Permi P (2011) Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons. J Biol Chem 286:2675–2680

    Article  PubMed  CAS  Google Scholar 

  19. Parkash V, Lindholm P, Peränen J, Kalkkinen N, Oksanen E, Saarma M, Leppänen VM, Goldman A (2009) The structure of the conserved neurotrophic factors MANF and CDNF explains why they are bifunctional. Protein Eng Des Sel 22:233–241

    Article  PubMed  CAS  Google Scholar 

  20. Lindholm P, Saarma M (2010) Novel CDNF/MANF family of neurotrophic factors. Dev Neurobiol 70:360–371

    PubMed  CAS  Google Scholar 

  21. Hoseki J, Sasakawa H, Yamaguchi Y, Maeda M, Kubota H, Kato K, Nagata K (2010) Solution structure and dynamics of mouse ARMET. FEBS Lett 584:1536–1542

    Article  PubMed  CAS  Google Scholar 

  22. Voutilainen MH, Bäck S, Pörsti E, Toppinen L, Lindgren L, Lindholm P, Peränen J, Saarma M, Tuominen RK (2009) Mesencephalic astrocyte-derived neurotrophic factor is neurorestorative in rat model of Parkinson’s disease. J Neurosci 29:9651–9659

    Article  PubMed  CAS  Google Scholar 

  23. Airavaara M, Shen H, Kuo CC, Peränen J, Saarma M, Hoffer B, Wang Y (2009) Mesencephalic astrocyte-derived neurotrophic factor reduces ischemic brain injury and promotes behavioral recovery in rats. J Comp Neurol 515:116–124

    Article  PubMed  Google Scholar 

  24. Yu YQ, Liu LC, Wang FC, Liang Y, Cha DQ, Zhang JJ, Shen YJ, Wang HP, Fang S, Shen YX (2010) Induction profile of MANF/ARMET by cerebral ischemia and its implication for neuron protection. J Cereb Blood Flow Metab 30:79–91

    Article  PubMed  CAS  Google Scholar 

  25. Oh-hashi K, Kunieda K, Hirata Y, Kiuchi K (2011) Biosynthesis and secretion of mouse cysteine-rich with EGF-like domains 2. FEBS Lett 585:2481–2487

    Article  PubMed  CAS  Google Scholar 

  26. Aridor M, Fish KN, Bannykh S, Weissman J, Roberts TH, Lippincott-Schwartz J, Balch WE (2001) The Sar1 GTPase coordinates biosynthetic cargo selection with endoplasmic reticulum export site assembly. J Cell Biol 152:213–229

    Article  PubMed  CAS  Google Scholar 

  27. Oh-hashi K, Koga H, Ikeda S, Shimada K, Hirata Y, Kiuchi K (2009) CRELD2 is a novel endoplasmic reticulum stress-inducible gene. Biochem Biophys Res Commun 387:504–510

    Article  PubMed  CAS  Google Scholar 

  28. Sawada M, Sun W, Hayes P, Leskov K, Boothman DA, Matsuyama S (2003) Ku70 suppresses the apoptotic translocation of Bax to mitochondria. Nat Cell Biol 5:320–329

    Article  PubMed  CAS  Google Scholar 

  29. Aravind L, Koonin EV (2000) SAP—a putative DNA-binding motif involved in chromosomal organization. Trends Biochem Sci 25:112–114

    Article  PubMed  CAS  Google Scholar 

  30. Walker JR, Corpina RA, Goldberg J (2001) Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature 412:607–614

    Article  PubMed  CAS  Google Scholar 

  31. Bruhn H (2005) A short guided tour through functional and structural features of saposin-like proteins. Biochem J 389:249–257

    Article  PubMed  CAS  Google Scholar 

  32. Oh-hashi K, Ito M, Tanaka T, Hirata Y, Kiuchi K (2009) Biosynthesis, processing, and secretion of glial cell line-derived neurotrophic factor in astroglial cells. Mol Cell Biochem 323:1–7

    Article  PubMed  CAS  Google Scholar 

  33. Sato K, Nakano A (2007) Mechanisms of COPII vesicle formation and protein sorting. FEBS Lett 581:2076–2082

    Article  PubMed  CAS  Google Scholar 

  34. Glembotski CC (2011) Functions for the cardiomyokine, MANF, in cardioprotection, hypertrophy and heart failure. J Mol Cell Cardiol 51:512–517

    Article  PubMed  CAS  Google Scholar 

  35. Zhang L, Lai E, Teodoro T, Volchuk A (2009) GRP78, but not protein-disulfide isomerase, partially reverses hyperglycemia-induced inhibition of insulin synthesis and secretion in pancreatic β-cells. J Biol Chem 284:5289–5298

    Article  PubMed  CAS  Google Scholar 

  36. Kudo T, Okumura M, Imaizumi K, Araki W, Morihara T, Tanimukai H, Kamagata E, Tabuchi N, Kimura R, Kanayama D, Fukumori A, Tagami S, Okochi M, Kub M, Tanii H, Tohyama M, Tabira T, Takeda M (2006) Altered localization of amyloid precursor protein under endoplasmic reticulum stress. Biochem Biophys Res Commun 344:525–530

    Article  PubMed  CAS  Google Scholar 

  37. Qian Y, Zheng Y, Weber D, Tiffany-Castiglioni EA (2007) 78-kDa glucose-regulated protein is involved in the decrease of interleukin-6 secretion by lead treatment from astrocytes. Am J Physiol Cell Physiol 293:C897–C905

    Article  PubMed  CAS  Google Scholar 

  38. Susuki S, Sato T, Miyata M, Momohara M, Suico MA, Shuto T, Ando Y, Kai H (2009) The endoplasmic reticulum-associated degradation of transthyretin variants is negatively regulated by BiP in mammalian cells. J Biol Chem 284:8312–8321

    Article  PubMed  CAS  Google Scholar 

  39. Munro S, Pelham HR (1987) A C-terminal signal prevents secretion of luminal ER proteins. Cell 48:899–907

    Article  PubMed  CAS  Google Scholar 

  40. Appenzeller-Herzog C, Ellgaard L (2008) The human PDI family: versatility packed into a single fold. Biochim Biophys Acta 1783:535–548

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Dr. Wei Liu and Dr. Jennifer Lippincott-Schwartz for providing HA-tagged Sar1 constructs (H79G). A part of this work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Young Scientists (B), No. 21700403 to K.O.).

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Correspondence to Kentaro Oh-hashi.

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Oh-hashi, K., Tanaka, K., Koga, H. et al. Intracellular trafficking and secretion of mouse mesencephalic astrocyte-derived neurotrophic factor. Mol Cell Biochem 363, 35–41 (2012). https://doi.org/10.1007/s11010-011-1155-0

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