Distinct dedifferentiation processes affect caveolin-1 expression in hepatocytes

Abstract

Background: Dedifferentiation and loss of hepatocyte polarity during primary culture of hepatocytes are major drawbacks for metabolic analyses. As a prominent profibrotic cytokine and potent inducer of epithelial mesenchymal transition (EMT), TGF-β contributes to these processes in liver epithelial cells. Yet, a distinction between culture dependent and TGF-β driven hepatocyte dedifferentiation has not been shown to date.

Results: Here, we show that in both settings, mesenchymal markers are induced. However, upregulation of Snai1 and downregulation of E-Cadherin are restricted to TGF-β effects, neglecting a full EMT of culture dependent hepatocyte dedifferentiation. Mechanistically, the latter is mediated via FAK/Src/ERK/AKT pathways leading to the induction of the oncogene caveolin-1 (Cav1). Cav1 was recently proposed as a new EMT marker, but our results demonstrate Cav1 is not up-regulated in TGF-β mediated hepatocyte EMT, thus limiting validity of its use for this purpose. Importantly, marking differences on Cav1 expression exist in HCC cell lines. Whereas well differentiated HCC cell lines exhibit low and inducible Cav1 protein levels - by TGF-β in a FAK/Src dependent manner, poorly differentiated cell lines display high Cav1 expression levels which are not further modulated by TGF-β.

Conclusions: This study draws a detailed distinction between intrinsic and TGF-β mediated hepatocyte dedifferentiation and elucidates cellular pathways involved. Additionally, by evaluating the regulation of the oncogene Cav1, we provide evidence to argue against Cav1 as a reliable EMT marker.

Figure 1

Intrinsic hepatocyte dedifferentiation is accompanied with upregulation of mesenchymal markers. (A) Time course of cultured hepatocytes stained with phalloidin. Starting on day 2, hepatocytes loose polar structure. Lower panel shows cells treated with TGF-β1 leading to similar actin remodeling and reduced cell-cell contacts over time. (B) Western blot showing a time course of murine hepatocytes cultured in monolayer on a stiff collagen matrix. Mesenchymal markers are induced and AKT and ERK signaling pathways are activated over time. Caveolin-1 is strongly upregulated during culture. (C) Collagen 1α1 (p < 0.001 at d3 and d4) and (D) caveolin-1 mRNA (p < 0.001 at d3 and d4) are upregulated in culture over time.

Figure 2

FAK activation influences caveolin-1 induction. (A) FAK and Src phosphorylation increases over time in culture. Blocking FAK phosphorylation with PF573228 (4 μM) culminates in reduced phospho-Src and N-Cadherin expression as well as attenuated caveolin-1 upregulation. E-Cadherin expression is unchanged. Caveolin-1 expression was densitometrically analyzed. A significant downregulation was observed at day 3 (p = 0.002) and day 4 (p = 0.011) (B) Blocking FAK (PF573228, 4 μM) leads to reduced AKT and ERK phosphorylation. Inhibition of Src kinases results in attenuated AKT and ERK phosphorylation. SU6656 (15 μM) additionally affects FAK phosphorylation, whereas PP2 (10 μM) does not. These data indicate that FAK signals via Src/ERK/AKT.

Figure 3

Src is a critical mediator for caveolin-1 induction. (A) SU6656 (SU; 15 μM) was used to block Src activity. Blockage yielded in reduced caveolin-1 protein (evaluated with densitometry) similar to (B) where PP2 (10 μM) was used. (C, D) Caveolin-1 mRNA expression is also reduced during culture in presence of inhibitors (p = 0.002 for SU6656 at d4; p < 0.001 for PP2 at d4). (E, F) Induction of Collagen 1α1 was reduced upon Src inhibition with both inhibitors (SU6656: p < 0.001; PP2: p = 0.005).

Figure 4

The AKT and ERK pathways are required for caveolin-1 induction. (A) Blocking AKT activation with Ly294002 decreased caveolin-1 upregulation on protein and (B) mRNA level (p = 0.018). (C) Induction of Collagen 1α1 mRNA, a mesenchymal marker, was completely abolished when AKT signaling was blocked (P < 0.001). (D) Inhibition of ERK activation via the small molecule inhibitor U0126 (U5: 5 μM; U10: 10 μM) culminated in strongly reduced caveolin-1 induction on protein and (E) mRNA level (p < 0.001). (F) ERK affects upregulation of Collagen 1α1 (p < 0.001) for mRNA expression studies, U0126 was used at a conc. of 10 μM.

Figure 5

The Snai1 family is not involved in intrinsic dedifferentiation and caveolin-1 upregulation. (A) Snai1 is slightly upregulated during dedifferentiation and tremendously and time dependently induced by TGF-β1 stimulation (**p < 0.005 at all time points when comparing untreated with TGF-β1; p < 0.05 untreated d3 vs. untreated d1). (B) No change of Snai2 (Slug) expression could be detected in hepatocytes cultured for 4 days. Beginning on day 3, TGF-β did increase its expression (p ≤ 0.002 at d3 and d4 comparing expression of untreated vs. TGF-β1). (C) Dedifferentiating hepatocytes from wild type and Snai1 ko mice were analyzed for dedifferentiation events with Western blot. No alterations in ERK activation and caveolin-1 induction were observed. (D) Proof of hepatocyte specific Snai1 knockout. Basal Snai1 expression was very weak in wild type hepatocytes (based on qPCR data) and almost absent in Snai1 ko hepatocytes. Induction of Snai1 expression was triggered by TGF-β1 stimulation for 6 h. Control hepatocytes responded with strong induction, whereas Snai1 knockout hepatocytes did not induce expression (p < 0.001).

Figure 6

Effects of TGF-β1 on caveolin-1 induction. (A) TGF-β1 treated hepatocytes display attenuated caveolin-1 protein upregulation. TGF-β1 dependent EMT induction is reflected by E-Cadherin downregulation. (B) Similar to the protein regulation, mRNA levels of caveolin-1 are less induced compared to corresponding controls (p ≤ 0.001 after 2, 3 and 4 days). (C) Knockdown of Smad4 attenuates TGF-β1 mediated caveolin-1 repression. (D) Inhibition of Src using SU6656 (15 μM) blocked intrinsic and TGF-β mediated upregulation of N-Cadherin and caveolin-1.

Figure 7

HCC cell lines and caveolin-1 expression. (A) HUH-7, Hep3B and PLC/PRF/5 are weak caveolin-1 expressing cells (both on mRNA and protein level, qPCR data normalized to 18S rRNA), whereas the dedifferentiated lines HLE, HLF and FLC-4 demonstrate high expression levels. (B) Based on elevated basal migration capacity, assessed with the transwell assay, HLE, HLF and FLC-4 are considered dedifferentiated (see text for details).

Figure 8

TGF-β induces caveolin-1 expression in a subset of HCC cell lines. (A) qReal-time analysis of TGF-β1 treated HCC cell lines. The low caveolin-1 expressing lines respond to TGF-β via an upregulation of expression (*p < 0.05; **p < 0.01), whereas HLE, HLF and FLC-4 do not change expression upon TGF-β1 challenge. (B) Western blot of PLC/PRF/5 lysates revealed caveolin-1 upregulation after 12 and 24 h of TGF-β1 treatment. (C) HUH-7 cells require 36 h TGF-β1 treatment to enhance caveolin-1 protein expression. (D) Hep3B cells strongly elevate caveolin-1 expression after 24 h of TGF-β1 treatment. Blocking FAK or Src phosphorylation by applying the chemical inhibitors PF573228 (4 μM) or SU6656 (15 μM) abolished the induction of caveolin-1 protein expression upon TGF-β1 stimulation.