Integrin expression and function in the response of primary culture hepatic stellate cells to connective tissue growth factor (CCN2)

Abstract

Production of connective tissue growth factor (CCN2, also known as CTGF) is a hallmark of hepatic fibrosis. This study examined early primary cultures of hepatic stellate cells (HSC) for (i) CCN2 regulation of its cognate receptor integrin subunits; and (ii) interactions between CCN2 and integrin α(5)β(1), heparan sulphate proteoglycans (HSPG) or fibronectin (FN) in supporting cell adhesion. HSC were isolated from healthy male Balb/c mice. mRNA levels of CCN2 or α(5), β(1), αv or β(3) integrin subunits were measured in days 1-7 primary culture HSC, and day 3 or day 7 cells treated with recombinant CCN2 or CCN2 small interfering RNA. Interactions between CCN2 and integrin α(5)β(1), HSPG or FN were investigated using an in vitro cell adhesion assay. Co-incident with autonomous activation over the first 7 days, primary culture HSC increasingly expressed mRNA for CCN2 or integrin subunits. Addition of exogenous CCN2 or knockdown of endogenous CCN2 differentially regulated integrin gene expression in day 3 versus day 7 cells. Either full length CCN2 ('CCN2(1-4)') or residues 247-349 containing module 4 alone ('CCN2(4)') supported day 3 cell adhesion in an integrin α(5)β(1) - and HSPG-dependent fashion. Adhesion of day 3 cells to FN was promoted in an integrin α(5) β(1)-dependent manner by CCN2(1-4) or CCN2(4), whereas FN promoted HSPG-dependent HSC adhesion to CCN2(1-4) or CCN2(4). These findings suggest CCN2 regulates integrin expression in primary culture HSC and supports HSC adhesion via its binding of cell surface integrin α(5)β(1), a novel CCN2 receptor in primary culture HSC which interacts co-operatively with HSPG or FN.

fig 1

α-SMA production by primary culture mHSC. (A) RT-PCR of total cellular RNA extracted from primary mHSC for determination of α-SMA gene expression on day 1, day 4 or day 7 of primary culture. A DNA marker ladder and non-cDNA template (NTC) are also shown. (B) Immunofluorescent detection of α-SMA protein in day 3 mHSC (magnification ×100). This staining pattern was seen in about 40% of the cells on day 3. (C) Quantitative real-time PCR detection of α-SMA mRNA on days 1–7. Data are the means ± S.D. of triplicate determinations and are representative of three independent experiments. *P < 0.05 versus day 1.

fig 2

CCN2 expression by primary culture mHSC. (A) RT-PCR of total cellular RNA extracted from primary mHSC for determination of CCN2 gene expression on day 1, day 4 or day 7 of primary culture. A DNA marker ladder and non-cDNA template (NTC) are also shown. (B) Quantitative real-time PCR detection of CCN2 mRNA on days 1–7. Data are the means ± S.D. of triplicate determinations and are representative of three independent experiments. *P < 0.05 versus day 1.

fig 3

Integrin expression by primary culture mHSC. Total cellular RNA extracted from primary cultures of mHSC was subjected to reverse transcription, followed by PCR (A, B) or real-time PCR (C) to determine gene expression of the α₅, αv, β₁ or β₃ intergin subunits. (A) RT-PCR for integrin α₅ assessed on day 1, day 4 or day 7. A DNA marker ladder and non-cDNA template (NTC) are also shown. (B) RT-PCR for integrin αv (lane 1), β₁ (lane 3) or β₃ (lane 5) and their non-cDNA template controls (lanes 2, 4 and 6, respectively). A DNA marker ladder is also shown. (C) Quantitative real-time PCR detection of integrins α₅, αv, β₁ or β₃ on days 1–7. Data are the means ± S.D. of triplicate determinations and are representative of three independent experiments. *P < 0.05 versus day 1.

fig 4

Regulation of integrin subunit expression by recombinant CCN2. mHSC on (A) day 3 or (B) day 7, both of which were pre-incubated in DMEM/F12 containing 0.5% foetal bovine serum for 24 hrs, were treated with or without CCN2 (200 ng/ml) for an additional 24 hrs prior to analysis of integrin gene expression by quantitative real-time PCR. The data are the means ± S.D. of triplicate determinations and are representative of three independent experiments. Ctrl: non-treated cells. *P < 0.05 versus Ctrl.

fig 5

CCN2 siRNA-mediated down-regulation of integrin expression. mHSC on (A) day 3 or (B) day 7 were treated for 48 hrs with siCCN2 (50 nM) prior to analysis of mRNA expression of the integrin α₅, αv, β₁ or β₃ subunits by quantitative real-time PCR. Data are the means ± S.D. of triplicate determinations and are representative of three independent experiments. *P < 0.05 versus Ctrl. (C) Immunofluorescent detection of CCN2 in day 1 (without siCCN2 treatment), day 3 or day 7 mHSC treated with or without siCCN2 (magnification ×100). NC: negative control.

fig 6

CCN2 or FN promotes mHSC adhesion. Day 3 mHSC were pre-incubated with 10 mM NaClO₃ in the presence or absence of 10 mM Na₂SO₄, for 24 hrs or with 10 μg/ml anti-integrin α₅β₁ antibody for 30 min. and then plated for 30 min. into 96-well tissue culture plates (10⁵ cells/well in 100 μl of medium) that had been pre-coated with 2 μg/ml CCN2_1–4, CCN2₄ or FN. Wells were then washed and the frequency of adherent cells was evaluated using a CyQuant NF Assay Kit. The data are the means ± S.D. of quadruplicate determinations and are representative of three independent experiments. *P < 0.05 versus BSA-coated group; ^#P < 0.05 versus Ctrl or NaClO₃+ Na₂SO₄ group; ^▴P < 0.05 versus Ctrl group.

fig 7

CCN2 promotes mHSC cell adhesion to FN. Day 3 primary mHSC were pre-incubated with or without 10 μg/ml anti-integrin α₅β₁ for 30 min. and then placed for 30 min. in 96-well tissue culture plates (10⁵ cells/well in 100 μl of medium) pre-coated with FN (2 μg/ml) in the presence or absence of 200 ng/ml CCN2_1–4 or CCN2₄. Wells were washed and the frequency of adherent cells was evaluated using a CyQuant NF Assay Kit. Data are representative of three separate experiments. Values represent mean ± S.D. of quadruplicate determinations from three experiments. *P < 0.05 versus Ctrl group; ^#P < 0.05 versus IgG group.

fig 8

FN promotes mHSC adhesion to CCN2. Day 3 primary mHSC were pre-incubated with or without 2 μg/ml heparin or 2 units/ml heparinase I in the presence or absence of 1× Protease Inhibitor Cocktail for 30 min., and then placed for 30 min. in 96-well tissue culture plates plates (10⁵ cells/well in 100 μl of medium) that had been pre-coated with 2 μg/ml CCN2_1–4 or CCN2₄, in the presence or absence of 200 ng/ml FN. Wells were washed and frequency of adherent cells was evaluated using a CyQuant NF Assay Kit. Data are representative of three separate experiments. Values represent mean ± S.D. of quadruplicate determinations from three experiments. Cocktail: protease inhibitor cocktail. *P < 0.05 versus Ctrl group; ^#P < 0.05 versus FN group; ^▴P < 0.05 versus FN group; **P > 0.05 versus Heparinase I group.