Journal of Molecular Neuroscience

, Volume 68, Issue 1, pp 135–143 | Cite as

Tuftelin Is Required for NGF-Induced Differentiation of PC12 Cells

  • Dekel ShiloEmail author
  • Gadi Cohen
  • Anat Blumenfeld
  • Koby Goren
  • Salem Hanhan
  • Shay Sharon
  • Amir Haze
  • Dan Deutsch
  • Philip Lazarovici


Nerve growth factor (NGF) promotes pleiotropic gene transcription-dependent biological effects, in neuronal and non-neuronal cells, including survival, proliferation, differentiation, neuroprotection, pain, and angiogenesis. It is hypothesized that during odontogenesis, NGF may be implicated in morphogenetic and mineralization events by affecting proliferation and/or differentiation of dental cells. Tuftelin belongs to the enamel associated teeth proteins and is thought to play a role in enamel mineralization. We previously reported that tuftelin transcript and protein, which are ubiquitously expressed in various tissues of embryos, adults, and tumors, were significantly upregulated during NGF-induced PC12 differentiation. To further confirm the involvement of tuftelin in the differentiation process, we established a tuftelin-knockdown neuronal PC12 cell model, using a non-cytotoxic siRNA directed towards sequences at the 3′ UTR of the tuftelin gene. Using real-time PCR, we quantified tuftelin mRNA expression and found that tuftelin siRNA, but not scrambled siRNA or transfection reagents, efficiently depleted about 60% of NGF-induced tuftelin mRNA transcripts. The effect of tuftelin siRNA was quantified up to 6 days of NGF-induced differentiation. Using immunofluorescence and western blot analyses, we also found a direct correlation between reduction of 60–80% in tuftelin protein expression and inhibition of about 50–70% in NGF-induced differentiation of the cells, as was detected after 3–6 days of treatment. These results demonstrate an important role for tuftelin in NGF-induced differentiation of PC12 cells. Tuftelin could be a useful target for drug development in disease where neurotrophin therapy is required.


Nerve growth factor NGF PC12 cells Cell differentiation Neuronal outgrowth Tuftelin Tuft1 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Aloe L, Luisa Rocco M, Omar Balzamino B, Micera A (2015) Nerve growth factor: a focus on neuroscience and therapy. Curr Neuropharmacol 13:294–303CrossRefGoogle Scholar
  2. Amano O, Bringas P, Takahashi I, Takahashi K, Yamane A, Chai Y, Nuckolls GH, Shum L, Slavkin HC (1999) Nerve growth factor (NGF) supports tooth morphogenesis in mouse first branchial arch explants. Dev Dyn 216:299–310CrossRefGoogle Scholar
  3. Angelastro JM, Klimaschewski L, Tang S, Vitolo OV, Weissman TA, Donlin LT, Shelanski ML, Greene LA (2000) Identification of diverse nerve growth factor-regulated genes by serial analysis of gene expression (SAGE) profiling. Proc Natl Acad Sci 97:10424–10429CrossRefGoogle Scholar
  4. Arien-Zakay H, Lecht S, Bercu MM, Amariglio N, Rechavi G, Galski H, Lazarovici P, Nagler A (2009) Interferon-γ-induced neuronal differentiation of human umbilical cord blood-derived progenitors. Leukemia 23:1790–1800CrossRefGoogle Scholar
  5. Cho K, Skarnes W, Minsk B, Palmieri S, Jackson-Grusby L, Wagner J (1989) Nerve growth factor regulates gene expression by several distinct mechanisms. Mol Cell Biol 9:135–143CrossRefGoogle Scholar
  6. Chung J, Kubota H, Y-i O, Uda S, Kuroda S (2010) Timing-dependent actions of NGF required for cell differentiation. PLoS One 5:e9011CrossRefGoogle Scholar
  7. D’Onofrio M, Paoletti F, Arisi I, Brandi R, Malerba F, Fasulo L, Cattaneo A (2011) NGF and proNGF regulate functionally distinct mRNAs in PC12 cells: an early gene expression profiling. PLoS One 6:e20839CrossRefGoogle Scholar
  8. Delgado S, Deutsch D, Sire J (2017) Evolutionary analysis of the mammalian tuftelin sequence reveals features of functional importance. J Mol Evol 84:214–224Google Scholar
  9. Deutsch D, Palmon A, Fisher LW, Kolodny N, Termine JD, Young MF (1991) Sequencing of bovine enamelin (“tuftelin”) a novel acidic enamel protein. J Biol Chem 266:16021–16028Google Scholar
  10. Deutsch D, Dafni L, Palmon A, Hekmati M, Young MF, Fisher LW (1997) Tuftelin: enamel mineralization and amelogenesis imperfecta. CIBA Found Symp 205:135–147 discussion 147-155Google Scholar
  11. Deutsch D, Palmon A, Dafni L, Mao Z, Leytin V, Young M, Fisher LW (1998) Tuftelin--aspects of protein and gene structure. Eur J Oral Sci 106(1):315–323Google Scholar
  12. Deutsch D et al (2002) The human tuftelin gene and the expression of tuftelin in mineralizing and nonmineralizing tissues. Connect Tissue Res 43:425–434Google Scholar
  13. Deutsch D, Silverstein N, Shilo D, Lecht S, Lazarovici P, Blumenfeld A (2011) Biphasic influence of hypoxia on tuftelin expression in mouse mesenchymal C3H10T1/2 stem cells. Eur J Oral Sci 119(Suppl 1):55–61CrossRefGoogle Scholar
  14. Dijkmans TF, van Hooijdonk LWA, Schouten TG, Kamphorst JT, Vellinga ACA, Meerman JHN, Fitzsimons CP, de Kloet ER, Vreugdenhil E (2008) Temporal and functional dynamics of the transcriptome during nerve growth factor-induced differentiation. J Neurochem 105:2388–2403CrossRefGoogle Scholar
  15. Dutta P, Koch A, Breyer B, Schneider H, Dittrich-Breiholz O, Kracht M, Tamura T (2011) Identification of novel target genes of nerve growth factor (NGF) in human mastocytoma cell line (HMC-1 (V560G c-Kit)) by transcriptome analysis. BMC Genomics 12:196CrossRefGoogle Scholar
  16. Fujita K, Lazarovici P, Guroff G (1989) Regulation of the differentiation of PC12 pheochromocytoma cells. Environ Health Perspect 80:127–142CrossRefGoogle Scholar
  17. Greene LA, Angelastro JM (2005) You can’t go home again: transcriptionally driven alteration of cell signaling by NGF. Neurochem Res 30:1347–1352CrossRefGoogle Scholar
  18. Katzir I, Shani J, Regev K, Shabashov D, Lazarovici P (2002) A quantitative bioassay for nerve growth factor, using PC12 clones expressing different levels of trkA receptors. J Mol Neurosci 18:251–264CrossRefGoogle Scholar
  19. Lazarovici P, Marcinkiewicz C, Lelkes PI (2006) Cross talk between the cardiovascular and nervous systems: neurotrophic effects of vascular endothelial growth factor (VEGF) and angiogenic effects of nerve growth factor (NGF)-implications in drug development. Curr Pharm Des 12:2609–2622CrossRefGoogle Scholar
  20. Leiser Y, Blumenfeld A, Haze A, Dafni L, Taylor AL, Rosenfeld E, Fermon E, Gruenbaum-Cohen Y, Shay B, Deutsch D (2007) Localization, quantification, and characterization of tuftelin in soft tissues. Anat Rec 290:449–454CrossRefGoogle Scholar
  21. Leiser Y, Silverstein N, Blumenfeld A, Shilo D, Haze A, Rosenfeld E, Shay B, Tabakman R, Lecht S, Lazarovici P, Deutsch D (2011) The induction of tuftelin expression in PC12 cell line during hypoxia and NGF-induced differentiation. J Cell Physiol 226:165–172CrossRefGoogle Scholar
  22. Leonard D, Ziff E, Greene L (1987) Identification and characterization of mRNAs regulated by nerve growth factor in PC12 cells. Mol Cell Biol 7:3156–3167CrossRefGoogle Scholar
  23. Levi A, Biocca S, Cattaneo A, Calissano P (1989) The mode of action of nerve growth factor in PC12 cells. In: Molecular neurobiology 1988. Springer, pp 201-226Google Scholar
  24. Lin JY-S, Wu CL, Liao CN, Higuchi A, Ling Q-D (2016) Chemogenomic analysis of neuronal differentiation with pathway changes in PC12 cells. Mol BioSyst 12:283–294CrossRefGoogle Scholar
  25. Mao Z, Shay B, Hekmati M, Fermon E, Taylor A, Dafni L, Heikinheimo K, Lustmann J, Fisher LW, Young MF, Deutsch D (2001) The human tuftelin gene: cloning and characterization. Gene 279:181–196CrossRefGoogle Scholar
  26. Mitsiadis TA, Pagella P (2016) Expression of nerve growth factor (NGF), TrkA, and p75(NTR) in developing human fetal teeth. Front Physiol 7:338Google Scholar
  27. Nosrat I, Seiger A, Olson L, Nosrat CA (2002) Expression patterns of neurotrophic factor mRNAs in developing human teeth. Cell Tissue Res 310:177–187CrossRefGoogle Scholar
  28. Rudkin B, Lazarovici P, Levi B, Abe Y, Fujita K, Guroff G (1989) Cell cycle-specific action of nerve growth factor in PC12 cells: differentiation without proliferation. EMBO J 8:3319–3325CrossRefGoogle Scholar
  29. Shay B, Gruenbaum-Cohen Y, Tucker AS, Taylor AL, Rosenfeld E, Haze A, Dafni L, Leiser Y, Fermon E, Danieli T, Blumenfeld A, Deutsch D (2009) High yield expression of biologically active recombinant full length human tuftelin protein in baculovirus-infected insect cells. Protein Expr Purif 68:90–98CrossRefGoogle Scholar
  30. Skaper SD (2017) Nerve growth factor: a neuroimmune crosstalk mediator for all seasons. Immunology 151:1–15CrossRefGoogle Scholar
  31. Tabakman R, Lazarovici P, Kohen R (2002) Neuroprotective effects of carnosine and homocarnosine on pheochromocytoma PC12 cells exposed to ischemia. J Neurosci Res 68:463–469CrossRefGoogle Scholar
  32. Vaudry D, Stork P, Lazarovici P, Eiden L (2002) Signaling pathways for PC12 cell differentiation: making the right connections. Science 296:1648–1649CrossRefGoogle Scholar
  33. Vician L, Basconcillo R, Herschman HR (1997) Identification of genes preferentially induced by nerve growth factor versus epidermal growth factor in PC12 pheochromocytoma cells by means of representational difference analysis. J Neurosci Res 50:32–43CrossRefGoogle Scholar
  34. Yan Q, Johnson EM Jr (1988) An immunohistochemical study of the nerve growth factor receptor in developing rats. J Neurosci 8:3481–3498CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Dental Research Laboratory, Faculty of Dental Medicine, Institute of Dental SciencesThe Hebrew University of JerusalemJerusalemIsrael
  2. 2.Rambam Health Care CampusHaifaIsrael
  3. 3.School of Pharmacy, Institute for Drug Research, Faculty of MedicineThe Hebrew University of JerusalemJerusalemIsrael

Personalised recommendations