SILAC labeling coupled to shotgun proteomics analysis of membrane proteins of liver stem/hepatocyte allows to candidate the inhibition of TGF-beta pathway as causal to differentiation
- Claudia Montaldo†1,
- Carmine Mancone†1, 2,
- Alice Conigliaro2,
- Angela Maria Cozzolino2,
- Valeria de Nonno2 and
- Marco Tripodi1, 2Email author
© Montaldo et al.; licensee BioMed Central Ltd. 2014
Received: 23 October 2013
Accepted: 11 March 2014
Published: 15 March 2014
Despite extensive research on hepatic cells precursors and their differentiated states, much remains to be learned about the mechanism underlying the self-renewal and differentiation.
We apply the SILAC (stable isotope labeling by amino acids in cell culture) approach to quantitatively compare the membrane proteome of the resident liver stem cells (RLSCs) and their progeny spontaneously differentiated into epithelial/hepatocyte (RLSCdH). By means of nanoLC-MALDI-TOF/TOF approach, we identified and quantified 248 membrane proteins and 57 of them were found modulated during hepatocyte differentiation. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the most of membrane proteins found to be modulated are involved in cell-to-cell signaling/interaction pathways. Moreover, the upstream prediction analysis of proteins involved in cell-to-cell signaling and interaction unveiled that the activation of the mesenchymal to epithelial transition (MET), by the repression of TGFB1/Slug signaling, may be causal to hepatocyte differentiation.
Taken together, this study increases the understanding of the underlying mechanisms modulating the complex biological processes of hepatic stem cell proliferation and differentiation.
Stem/precursor cells are able to differentiate into almost every cell type and are therefore promising tools for the development of new therapeutic approaches in regenerative medicine . Much attention has focused on understanding stem cell biology and the regulation of differentiation to help realize these clinical aspirations [2, 3]. However, the key biochemical networks controlling the stem cell biology remain poorly described or undefined, contributing to a lack of knowledge of the principle events regulating differentiation processes.
Quantitative proteomics allows to gather unbiased wide range evidences instrumental to gain deeper insight into the regulatory networks controlling cellular biology [4, 5]. This is now possible by integrating proteomic data sets with literature databases . With respect to stem/precursor cells, characterization of molecular pathways causal for self-renewal and multi-step differentiation is desirable. Recent successful studies have allowed for addressing these issues in precursors of many histotypes including self-renewing and differentiating embryonic stem cells , neural stem cell differentiation toward astroglial  and primary cultured hepatocytes at different stages of development .
In this work, we compared the proteome profile of a stem/precursor cell directly with its differentiated progeny: resident liver stem cells (RLSCs) were compared to their progeny spontaneously differentiated into epithelial/hepatocyte (RLSCdH) .
RLSCs, previously isolated from murine liver explants, were shown to be non-tumorigenic multi-potent Sca1+ CD34-, CD45-, albumin- stem cell lines capable of i) self-renewal ii) spontaneous differentiation into hepatocytes and cholangiocytes iii), and inducible to differentiating into mesenchymal and neuro-ectodermal cell lineages such as osteoblasts/osteocytes, chondrocytes, astrocytes and neural cells when cultured in appropriate conditions .
Moreover, at later stages of differentiation, RLSCdH were shown to switch from hepatocytes bearing a periportal phenotype to perivenular hepatocytes in dependence to Wnt pathway activation . More recently, in orthotropic transplants, RLSCs have also been shown to generate epithelial and mesenchymal liver-specific derivatives (i.e. hepatocytes and hepatic stellate cells) properly integrated in the liver architecture .
Here, we aimed to identify RLSC functional elements underlying either self-renewal or differentiation toward hepatocytes. We therefore compared the membrane protein profile of self-renewing RLSCs with those of RLSCdH by using SILAC-based quantitative proteomics. The expression of 57 membrane-associated proteins was found differently regulated. We then systematically studied the differentially expressed membrane proteins. The data highlight the interaction networks and metabolic pathways involved in hepatic stem cell proliferation and differentiation.
Identification and quantification of membrane proteins of RLSCs and RLSCdH
Cell growth of stem cells is often a major obstacle for metabolically labeled-based quantitative proteomic analysis. Since RLSCs display long term self-renewing capability, they provide a suitable cellular model to address by SILAC-based proteomics studies of molecular mechanisms controlling stem cell maintenance and differentiation.
Membrane proteins were then separated by SDS-PAGE and the gel lane was cut into 16 sections ranging from 15 to 250 kDa. Proteins in each gel section were then digested and submitted to nanoLC-MALDI-TOF/TOF analysis. Proteins were considered identified when at least two peptides were found fragmented by MS/MS, and were considered differentially expressed when the SILAC ratio was Heavy/Light or Light/Heavy ≥1.5. These criteria allowed for listing a total of 57 proteins (~23% of the total proteins identified) (Additional file 1); 31 were found overexpressed in RLSCs and 26 were found overexpressed in RLSCdH. Interestingly, by comparing the membrane proteomic data set with that obtained by means of SILAC approach on the whole cell extracts, we found that differences in membrane-associated protein expression between RLSCs and RLSCdH was due not only by changes in total cellular expression but also as the result of a different cellular localization (Additional file 2).
Validation of the expression of selected cell surface proteins
Differentially expressed cell surface proteins during hepatocyte differentiation of RLSC
Fold change DH/RLSC
Bioinformatic analysis of the differentially expressed membrane proteins
Stem/precursor cell differentiation into epithelial cells and persistent maintenance of epithelial phenotype are processes tightly regulated by membrane protein signaling pathways [8, 13]. Here, by means of SILAC-based proteomic approach, we compared the membrane proteome of self-renewing RLSCs and their epithelial progeny RLSCdH. This allowed us to quantify a differential protein expression as well as, following computational biology approaches, predict signaling events causal to hepatocyte differentiation.
Among the proteins found to be overexpressed in the RLSCdH, we identify and quantify high levels of three proteins involved in cell adhesion: E-cadherin, integrin beta-4 and galectin-4. Since these proteins have been well-characterized as markers of epithelial polarity [14–16], our findings are in line with our previous report describing the epithelial features of RLSCdH .
Our proteomic data set has also been analyzed to unveil potential changes in molecular functions. Through IPA analysis, we found that hepatocyte differentiation leads to extensive changes in cell-to-cell signaling and interaction protein expression. These changes, when functionally correlated, revealed how cell surface-driven signaling may drive the differentiation of liver precursor cells. Interestingly, proteins involved in cell adhesion or motility (Figure 4), including E-cadherin, integrin beta-4, junction plakoglobin and vimentin, have been reported to be directly related to the MET [17–20]. This is consistent with previous reports stating that the activation of MET signaling regulates the balance between embryonic stem cell self-renewal and differentiation . The activation of MET and the reverse process of epithelial to mesenchymal transition (EMT) is regulated by the balance of different master genes. In particular, the liver-enriched transcription factors (LEFTs) like HNF4 are known to be master genes of epithelial/hepatocyte differentiation while the expression of members of the transcriptional repressors belonging to the Snail family regulates the induction of the EMT program and acquisition of stemness traits [22, 23]. Recently, it has emerged that members of these families are capable of directly repressing each other and regulating target genes involved in stemness and hepatocyte differentiation in an opposite manner . Here, by IPA up-stream analysis, we unveil that the inhibition of TGFβ, leading to the inhibition of SLUG transcriptional repressors belonging to the Snail family , could be responsible for MET activation in hepatocyte differentiation.
Quantitative SILAC measurement of membrane-associated proteins during the hepatocyte differentiation of RLSCs allowed to reveal signaling pathways activated during the hepatocyte differentiation process. In particular, these findings suggest a direct link between the mesenchymal to epithelial transition and the gain of epithelial properties during stem cell differentiation. This helps to gain insight into potential regulatory networks involved in both the maintenance of the hepatic stem cells multi-potent state and the processes of specific cell lineage differentiation.
Information on reagents and proteomic procedures are described in Additional file 4.
Cell culture and SILAC labeling
All cells were maintained on collagen coating dish at 37°C in 5% CO2. RLSC and RLSCdH cell lines were respectively grown in DMEM SILAC “heavy” (13C615N4-arginine and 13C6-lysine) and RPMI SILAC “light” (12C614N4-arginine and 12C6-lysine), supplemented with 10% fetal bovine serum (FBS) with recombinant human insulin (10 mg/ml), EGF (50 ng/ml), IGF II (30 ng/ml), for 8 passages before the experiment. This period lasted about 3 weeks, where the SILAC “heavy” cells’ labeling was complete.
Then, all cells were lysed and membrane proteins were isolated following the Membrane Protein Extraction Kit (M-PEK) protocol. Samples were analyzed by Bradford assay to determine the protein concentration, then dissolved in SDS-loading buffer and were analyzed by Western blotting. Equal amounts (155 μg) of membrane proteins from RLSC and RLSCdH cell lines were mixed and 100 μg of this were subsequently analyzed for proteomic analysis as described below. SILAC labeling and proteomic analysis were performed twice.
Protein digestion and peptide purification
100 μg of membrane proteins obtained from the SILAC experiment were separated on 4 − 12% gradient gels (Invitrogen), stained by Simply Blue Safe Stain staining and visualized. Sixteen sections of the gel lane were cut. Protein-containing gel pieces were washed with 100 μL of 0.1 M ammonium bicarbonate (5 min at RT). Then, 100 μL of 100% acetonitrile (ACN) was added to each tube and incubated for 5 min at RT. The liquid was discarded, the washing step repeated once more, and the gel plugs were shrunk by adding ACN. The dried gel pieces were reconstituted with 100 μL of 10 mM DTT/0.1 M ammonium bicarbonate and incubated for 40 min at 56°C for cysteine reduction. The excess liquid was then discarded and cysteines were alkylated with 100 μL of 55 mM IAA/0.1 M ammonium bicarbonate (20 min at RT, in the dark). The liquid was discarded, the washing step was repeated once more, and the gel plugs were shrunk by adding ACN. The dried gel pieces were reconstituted with 12.5 ng/μL trypsin in 50 mM ammonium bicarbonate and digested overnight at 37°C. The supernatant from the digestion was saved in a fresh tube and 100 μL of 1% TFA/30% ACN were added on the gel pieces for an additional extraction of peptides. The extracted solution and digested mixture were then combined and vacuum centrifuged for organic component evaporation. Peptides were resuspended with 40 μL of 2.5% ACN/0.1% TFA, desalted and filtered through a C18 microcolumn ZipTip, and eluted from the C18 bed using 10 μL of 80% ACN/0.1% TFA. The organic component was once again removed by evaporation in a vacuum centrifuge and peptides were resuspended in a suitable nanoLC injection volume (typically 3–10 μL) of 2.5% ACN/0.1% TFA.
Differentially expressed proteins were analyzed using Ingenuity Pathway Analysis (IPA, Ingenuity Systems; see http://www.ingenuity.com). The over-represented biological processes, molecular functions, and canonical pathways were generated based on information contained in the Ingenuity Pathways Knowledge Base. Right-tailed Fisher’s exact test was used to calculate a p-value determining the probability that each biological function and/or disease involved in that proteome profile alteration is due to chance alone.
Western blotting assay
Proteins were separated on Bis-Tris 4 − 12% gradient polyacrylamide gels (Invitrogen) and transferred on nitrocellulose membranes. Membranes were then blocked with 0.5% Tween-PBS containing 5% nonfat dried milk and incubated overnight with the primary antibodies followed by incubation with HRP-conjugated species-specific secondary antibodies and enhanced chemiluminescence reaction using ECL Plus Western Blotting Detection System.
We are deeply grateful to Ms. Andrea Baker (INMI Rome, Italy) for the editing.
This work was supported by grants from MIUR Ministero dell’Università e Ricerca Scientifica (FIRB 2012, codice progetto RBFR12NSCF), Associazione Italiana per la Ricerca sul Cancro (AIRC) and Ministero della Salute (Ricerca Corrente). Progetto Regione Lazio - Legge Regionale 26 del 28 Dicembre 2007 Art. 33 – comma 4 lettera b, title: “sviluppo di terapie cellulari alternative al trapianto di fegato mediante cellule staminali epatiche adulte”.
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