Proteomic profiling of the stem cell response to retinoic acid and synthetic retinoid analogues: identification of major retinoid-inducible proteins.
The natural retinoid, all-trans retinoic acid (ATRA), is widely used to direct the in vitro differentiation of stem cells. However, substantial degradation and isomerisation of ATRA in response to UV-vis light has serious implications with regard to experimental reproducibility and standardisation. We present the novel application of proteomic biomarker profiling technology to stem cell lysates to rapidly compare the differentiation effects of ATRA with those of two stable synthetic retinoid analogues, EC19 and EC23, which have both been shown to induce differentiation in the embryonal carcinoma cell line TERA2.cl.SP12. MALDI-TOF MS (matrix-assisted laser desorption ionisation time-of-flight mass spectrometry) protein profiles support previous findings into the functional relationships between these compounds in the TERA2.cl.SP12 line. Subsequent analysis of protein peak data enabled the semi-quantitative comparison of individual retinoid-responsive proteins. We have used ion exchange chromatographic protein separation to enrich for retinoid-inducible proteins, thereby facilitating their identification from SDS-PAGE gels. The cellular retinoid-responsive proteins CRABP-I, CRABP-II, and CRBP-I were up-regulated in response to ATRA and EC23, indicating a bona fide retinoid pathway response to the synthetic compound. In addition, the actin filament regulatory protein profilin-1 and the microtubule regulator stathmin were also elevated following treatment with both ATRA and EC23. The up-regulation of profilin-1 and stathmin associated with retinoid-induced neural differentiation correlates with their known roles in cytoskeletal reorganisation during axonal development. Immunological analysis via western blotting confirmed the identification of CRABP-I, profilin-1 and stathmin, and supported their observed regulation in response to the retinoid treatments.
Maltman
Daniel J
DJ
School of Biological and Biomedical Sciences, Durham University, Science Laboratories, South Road, Durham, UKDH1 3LE.
Christie
Victoria B
VB
Collings
Jonathan C
JC
Barnard
Jonathan H
JH
Fenyk
Stepan
S
Marder
Todd B
TB
Whiting
Andrew
A
Przyborski
Stefan A
SA
D001565:Benzoates / Q000494:pharmacology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D020869:Gene Expression Profiling; D040901:Proteomics / Q000379:methods*; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000378:metabolism; D054840:Retinol-Binding Proteins, Cellular / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism*; D013764:Tetrahydronaphthalenes / Q000494:pharmacology*; D014212:Tretinoin / Q000031:analogs & derivatives / Q000494:pharmacology*
D001565:Benzoates; C542238:EC19 compound; C542239:EC23 compound; D018168:Receptors, Retinoic Acid; D054840:Retinol-Binding Proteins, Cellular; D013764:Tetrahydronaphthalenes; C082927:retinoic acid binding protein I, cellular; C082928:retinoic acid binding protein II, cellular; D014212:Tretinoin