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, Carmine Mancone, Alice Conigliaro, Angela Maria

Cozzolino, Valeria de Nonno, Marco Tripodi

► To cite this version:

Claudia Montaldo, Carmine Mancone, Alice Conigliaro, Angela Maria Cozzolino, Valeria de Nonno, et al.. 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.. Proteome Science, 2014, 12 (1), pp.15. 10.1186/1477-5956-12-15. pasteur-01202583

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# 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<fnref n="1" />†, Carmine Mancone<fnref n="1" />,<fnref n="2" />†, Alice Conigliaro<fnref n="2" />, Angela Maria Cozzolino<fnref n="2" />, Valeria de Nonno<fnref n="2" /> and Marco Tripodi<fnref n="1" />,<fnref n="2" />*

**Abstract**

**Background:** 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.

**Results:** 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.

**Conclusions:** Taken together, this study increases the understanding of the underlying mechanisms modulating the complex biological processes of hepatic stem cell proliferation and differentiation.

**Keywords:** Stem cell, Hepatocyte, SILAC, Proteomics

**Background**

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 [1]. 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 [6]. 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 [7], neural stem cell differentiation toward astroglial [8] and primary cultured hepatocytes at different stages of development [9].

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

<figure><img src="image_3.png" /></figure>

<footnote>* Correspondence: tripodi@bce.uniroma1.it</footnote>

<footnote>†Equal contributors</footnote>

<footnote n="1">¹National Institute for Infectious Diseases L. Spallanzani, IRCCS, via Portuense 292, 00149 Rome, Italy</footnote>

<footnote n="2">²Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Cellular Biotechnologies and Haematology, Sapienza University of Rome, Via Regina Elena 324, 00161 Rome, Italy</footnote>
progeny spontaneously differentiated into epithelial/hep-
atocyte (RLSCdH) [10].

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 [10].

Moreover, at later stages of differentiation, RLSCdH
were shown to switch from hepatocytes bearing a peripor-
tal phenotype to perivenular hepatocytes in dependence to
Wnt pathway activation [11]. 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 [12].

Here, we aimed to identify RLSC functional elements
underlying either self-renewal or differentiation toward
hepatocytes. We therefore compared the membrane pro-
tein profile of self-renewing RLSCs with those of RLSCdH
by using SILAC-based quantitative proteomics. The expres-
sion of 57 membrane-associated proteins was found dif-
ferently regulated. We then systematically studied the
differentially expressed membrane proteins. The data high-
light the interaction networks and metabolic pathways in-
volved in hepatic stem cell proliferation and differentiation.

**Results**

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 ana-
lysis. Since RLSCs display long term self-renewing cap-
ability, they provide a suitable cellular model to address
by SILAC-based proteomics studies of molecular mecha-
nisms controlling stem cell maintenance and differentiation.

A self-renewing RLSCs population was long-term meta-
bolically labeled with ¹³C₆ ¹⁵N₄-arginine and ¹³C₆-lysine
(Heavy) while its differentiated cellular counterpart RLSCdH
was grown in light medium (Figures 1A and 1B). Whole
cell extracts were then isolated separately and equal
amounts of protein from each cell line were mixed and
subjected to native membrane purifications by means of
Membrane Protein Extraction Kit (M-PEK, Calbiochem).
As shown in Figure 1C, a highly enriched membrane
protein fraction was obtained. This is mainly due to
the membrane isolation kit used in the experiment. In
fact, in contrast to the two-phase partitioning technique,
where detergents are used to separate membrane proteins
based primarily on their intrinsic hydrophobicity, the
kit's scalable differential extraction procedure select-
ively extracts integral membrane and membrane-associated

proteins based on their actual association with cellular
membrane.

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 ana-
lysis. 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 over-
expressed 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 differ-
ences 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 cel-
lular localization (Additional file 2).

Validation of the expression of selected cell surface proteins

Among the 57 proteins quantified, proteins associated to
plasma membrane and with a measured fold of variation
higher than 2.0 were considered for validation (Table 1).
As shown in Figure 2, by means of western blotting
analysis we found that the membrane expression levels
of e-cadherin, integrin beta-4 and galectin-4 were similar
to those measured by quantitative proteomics.

Bioinformatic analysis of the differentially expressed
membrane proteins

In order to unveil the molecular functions underlying
RLSC differentiation, identified proteins were functionally
grouped according to the Ingenuity Pathways Analysis lit-
erature database. All the differentially expressed proteins
were therefore uploaded to the IPA server. Firstly, we ana-
lyzed our proteomic data set in the frame of molecular
and cellular functions. Cell-to-cell signaling/interaction,
energy production, lipid metabolism, small molecule bio-
chemistry, cellular movement, and cell death and survival
were the most significant listed functions, as determined
by the *p* value (Figure 3A). Interestingly, proteins involved in
the cell-to-cell signaling/interaction processes were listed as
a top-listed function (with a significant *p* value <0.000001)
while the tissue development was indicated as top-listed
(*p* < 0.000001) for physiological system development and
function, (Figure 3B).

Secondly, pathway analysis was also used to analyze
different functional networks. Four networks related to
previously described functions were generated. Particularly,
23 membrane proteins involved in cell-to-cell signaling
and interaction, tissue development and cellular movement
were grouped as top 1 network, (IPA score 46) (Figure 4),
<figure><img src="image_4.png" /></figure>

thus suggesting that these molecular and cellular functions orchestrate the differentiation of RLSCs.

Notably, we performed a further IPA analysis in cell-to-cell signaling and interaction to predict upstream molecules, including growth factors and transcription regulators, which may be causal to the observed protein expression

<table><caption>**Table 1** Differentially expressed cell surface proteins during hepatocyte differentiation of RLSC</caption><thead><tr><td>Accession number<sup>a</sup></td><td>Protein name</td><td>Fold change DH/RLSC</td><td>Function</td></tr></thead><tbody><tr><td>P09803</td><td>E-cadherin</td><td>3.6</td><td>Cell adhesion</td></tr><tr><td>A2A863</td><td>Integrin beta-4</td><td>2.6</td><td>Cell adhesion</td></tr><tr><td>Q8K419</td><td>Galectin-4</td><td>2.0</td><td>Cell adhesion</td></tr></tbody></table>

changes (Additional file 3). This analysis suggested that the expression of 16 of these proteins could be promoted by the inhibition of transforming growth factor beta 1 (TGFB1) and the transcriptional repressor Slug (SNAI2) (Figure 5). In fact, we observed up-regulation of E-cadherin (CDH1), Integrin alpha-3 (ITGA3), Integrin beta-4 (ITGB4), Junction plakoglobin (JUP) and down-regulation of vimentin (VIM); all these modulations are coherently related to mechanisms underlying the activation of mesenchymal to epithelial transition (MET) and in turn this transition requires the repression of TGFB1/Slug signaling.

## Discussion

Stem/precursor cell differentiation into epithelial cells and persistent maintenance of epithelial phenotype are

<footnote><sup>a</sup>According to Uniprot.</footnote>
<figure><img src="image_5.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_6.png" /></figure>

**Figure 2** Validation of the expression of selected cell surface proteins in RLSCs and DH. (A) Western blot analysis in total cell extract (WCE), soluble (S) and membrane (M) fractions for the indicated proteins. For each fraction, 10 µg of protein sample were loaded; GAPDH and Calnexin expressions were used respectively as total and membrane proteins loading control. One representative experiment out of three is shown. (B) Bands were analyzed by densitometry using Quality-One software (Bio-Rad laboratories, Richmond, CA). The Y axis shows the relative intensity respect to GAPDH (WCE) and calnexin (M). All data were from at least three independent experiments and shown as mean ± SD.

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 [11].

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 [21]. 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 [24]. 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 [25], could be responsible for MET activation in hepatocyte differentiation.

## Conclusions

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
<figure><img src="image_7.png" /></figure>

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.

## Methods

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” ($ ^{13}C_6 $ $ ^{15}N_4 $-arginine and $ ^{13}C_6 $-lysine) and RPMI SILAC “light” ($ ^{12}C_6 $ $ ^{14}N_4 $-arginine and $ ^{12}C_6 $-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
<figure><img src="image_8.png" /><figcaption>**Figure 4** Molecular pathway analysis of regulated membrane protein changes in RLSCs as they undergo hepatocytic differentiation.</figcaption></figure>

The shown network (Network 1) reveals protein interactions involved in cell-to-cell signaling and interaction, tissue development and cellular movement processes. Red represents an increase in protein expression, whereas green represents a decrease in expression level; the color intensity represents the degree of abundance change. A solid line indicates a direct interaction, and a dashed line indicates an indirect interaction.

<figure><img src="image_9.png" /><figcaption>**Figure 5** Upstream regulator analysis of membrane protein changes in differently regulated proteins involved in cell-to-cell signaling and interaction. Transforming growth factor beta 1 and the transcriptional factor Slug were identified with significant predicted activation score respectively of -2,20 and -2,22. Symbols are indicated in the predicted legend.</figcaption></figure>
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.

## Data analysis

Differentially expressed proteins were analyzed using Ingenuity Pathway Analysis (IPA, Ingenuity Systems; see 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.

## Additional files

Additional file 1: List of protein identification and SILAC-based quantitation on membrane-associated proteins.

Additional file 2: List of protein identification and SILAC-based quantitative on WCE-associated proteins. Comparative analysis between SILAC quantitation of membrane and WCE-associated proteins.

Additional file 3: List of upstream regulators of membrane protein changes in RLSCs when they undergo hepatocytic differentiation.

Additional file 4: Additional Methods.

## Competing interest

The authors declare that they have no competing interests.

## Authors' contributions

CIM and CM designed the study, carried out the cell cultures, performed and analyzed the proteomic analysis. MT, AC, VdN and AMC participated in the study design and analyzed the data. CM and MT conceived the study and drafted the manuscript. All authors have read and approved the final manuscript.

## Acknowledgment

We are deeply grateful to Ms. Andrea Baker (INMI Rome, Italy) for the editing.

## Financial support

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".

Received: 23 October 2013 Accepted: 11 March 2014

Published: 15 March 2014

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doi:10.1186/1477-5956-12-15

Cite this article as: Montaldo et al.: 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. *Proteome Science* 2014 *12*:15.

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