Proteomic profiling of the stem cell response to retinoic acid and synthetic retinoid analogues: identification of major retinoid-inducible proteins

Abstract

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.

Synthesis and applications of 2,4-disubstituted thiazole derivatives as small molecule modulators of cellular development

“…Reactions were monitored in situ by TLC, GC-M S or 1 H NM R spectroscopy to ensure consumption of starting materials before reaction workup. Thin layer chromatography (TLC) was performed on Polygram SIL G/UV254 plastic-backed silica gel 25 plates with visualisation achieved using a UV lamp. Column chromatography was performed with Davisil Silica gel, 60 mesh.…”

“…In order to minimise ATRA degradation and isomerisation, all culture flasks were handled under reduced light conditions. For reproducibility of results, cell cultures for each compound were set up in triplicate for each time point and 25 incubated for over 3, 7 and 14 day s. After 7 days, it became apparent that cell proliferation in cultures exposed to compound 3 had slowed considerably, which is consistent with cells committing to differentiate in response to ATRA. In contrast, cells treated with compounds 1 and 2 30 continued to proliferate, and very high cell numbers were observed ( Fig.…”

“…These small molecules are involved in regulating important biological pathways from embryogenesis through to adult homeostasis, and influence the proliferation and differentiation of a diverse range 25 of cell types, including those of the nervous system. 1 All-transretinoic acid (ATRA) in particular is the major metabolite of vitamin A; however, due to the presence of five conjugated double bonds, it is susceptible to photo-isomerisation into different retinoic acid isomers.…”

“…8 It may be postulated, therefore, that ligands with larger side-chains are able to occupy the additional space within the RARβ retinoid binding site and hence, may acquire selectivity for this subtype. 25 Differential promoter usage and alternative splicing produce the different isoforms observed with each RAR subtype; the RARβ gene has four isoforms: β1-4. RARβ2 is the most abundant 30 isoform and is of particular interest in the area of neuroscience research.…”

“…significant increase in antigen expression was observed in response to compounds 1 and 2, yet by day 14, an elevated A2B5 expression was recorded that was similar to the levels observed with ATRA and compound 3. It would appear that differentiated cell types were present in these cultures; however, further 25 investigation is required to determine if this was the result of spontaneous differentiation in the cultures or sub-optimal growth conditions rather than the effects induced by these compounds. Nevertheless, given the length of time required before a change in cell phenotype was observed, it is unlikely that compounds 1 and 30 2 behave as true inducers of differentiation.…”