Activation of JAK-STAT pathway is required for platelet-derived growth factor-induced proliferation of pancreatic stellate cells

Figure 1 Activated PSCs expressed PDGF β-receptor. A: Serum-starved, culture-activated PSCs were grown directly on slides. Immunostaining for PDGF β-receptor was performed using a streptavidin-biotin-peroxidase complex detection kit. Original magnification: ×20 objective; B: Serum-starved PSCs were treated with PDGF-BB (at 25 ng/mL) for the indicated time. Total cell lysates were prepared, and separated by 70 g/L SDS-polyacrylamide gel electrophoresis. The phosphorylation of PDGF β-receptor was examined by Western blotting.

Figure 2 PDGF activated STAT1 and STAT3. A: PSCs were treated with PDGF-BB (at 25 ng/mL, lane 2) in serum-free medium for 15 min. Nuclear extracts were prepared and subjected to electrophoretic mobility shift assay using STAT consensus oligonucleotide probe m67. Arrow denotes specific inducible complex competitive with cold double-stranded oligonucleotide probe (lane 3). For super shift assays, nuclear extracts were incubated with antibodies against STAT1 (lane 4) or STAT3 (lane 5) before incubation with the radiolabeled probe. *: super shifts. Lane 1: control (serum-free medium only); B: PSCs were treated with PDGF-BB (at 25 ng/mL) for the indicated time. Total cell lysates (approximately 100 μg) were prepared, and separated by 100 g/L SDS-polyacrylamide gel electrophoresis. The activation of STAT1 and STAT3 was examined by Western blotting using anti-phosphospecific antibodies. The levels of total STAT1 and STAT3 were also determined.

Figure 3 Src and JAK2 mediate the activation of STAT1 and STAT3. A: PSCs were treated with PDGF-BB (at 25 ng/mL) for the indicated time. Total cell lysates (approximately 100 μg) were prepared, and separated by 100 g/L SDS-polyacrylamide gel electrophoresis. The activation of Src and JAK2 was examined by Western blotting using anti-phosphospecific antibodies. The level of GAPDH was also determined; B-D: PSCs were treated with a Src inhibitor PP1 (at 1 or 2.5 μmol/L) or a JAK2 inhibitor AG490 (at 10 or 25 μmol/L) in the absence or presence of PDGF-BB (at 25 ng/mL) for 5 min. Total cell lysates (approximately 100 μg) were prepared, and the levels of phosphorylated JAK2, STAT1, STAT3, and PDGF β-receptor were determined by Western blotting. The levels of total STAT3 and GAPDH were also determined.

Figure 4 Ethanol and acetaldehyde decreased basal activation of JAK2 and STAT3. PSCs were treated with ethanol (at 50 mmol/L, panel A) or acetaldehyde (at 200 mmol/L, panel B) for the indicated time, or with PDGF-BB (at 25 ng/mL) for 5 min. Total cell lysates (approximately 100 μg) were prepared, and separated by 100 g/L SDS-polyacrylamide gel electrophoresis. The activation of JAK2, STAT1, and STAT3 was examined by Western blotting using anti-phosphospecific antibodies. The level of GAPDH was also determined.

Figure 5 Activation of Src and JAK2 is required for PDGF-induced proliferation and serum-starved PSCs were treated with PDGF-BB (at 25 ng/mL) in the presence or absence of AG490 (at 10 or 25 μmol/L) or PP1 (at 1 or 2. 5 μmol/L). After 24-h incubation, DNA synthesis was assessed by BrdU incorporation ELISA. Data are shown as mean±SD (n = 6). ^bP<0.01 versus PDGF-BB only. Con: control (serum-free medium only), A: optical density.

Figure 6 Activation of STAT3, and not STAT1, is required for PDGF-induced proliferation. A: PSCs were treated with antisense (“AS”) or sense (“SE”) oligonucleotides for STAT1 or STAT3 for 24 h. The expression of total STAT1 and STAT3 was examined by Western blotting; B: PSCs were treated with sense, antisense, or scramble (“SC”) oligonucleotides. After 24 h, cells were stimulated with PDGF-BB (at 25 ng/mL) for another 24 h, and DNA synthesis was assessed by BrdU incorporation enzyme-linked immunosorbent assay. Data are shown as mean±SD (% of the control, n = 6). ^bP<0.01 vs PDGF-BB only. Con: control (serum-free medium only), A: optical density.

Figure 7 Src, not JAK2, is located upstream of ERK. PSCs were treated with a JAK2 inhibitor AG490 (A) (at 10 or 25 μmol/L), a Src inhibitor PP1 (B) (at 1 or 2.5 μol/L), or a MAP kinase kinase inhibitor U0126 (C) (at 5 or 10 μmol/L) in the absence or presence of PDGF-BB (at 25 ng/mL) for 5 min. Total cell lysates were prepared, and the levels of phosphorylated ERK, STAT1, and STAT3 were determined by Western blotting. The level of total ERK was also determined.

Figure 8 Signaling pathways for PDGF-induced proliferation in PSCs.