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. 1999 Sep 28;96(20):11410-5.
doi: 10.1073/pnas.96.20.11410.

Platelet-derived growth factor beta receptor regulates interstitial fluid homeostasis through phosphatidylinositol-3' kinase signaling

R Heuchel  1 , A Berg, M Tallquist, K Ahlén, R K Reed, K Rubin, L Claesson-Welsh, C H Heldin, P Soriano
Affiliations

Platelet-derived growth factor beta receptor regulates interstitial fluid homeostasis through phosphatidylinositol-3' kinase signaling

R Heuchel et al. Proc Natl Acad Sci U S A. .

Abstract

Platelet-derived growth factor (PDGF) isoforms lead to mitogenic, survival, and chemotactic responses in a variety of mesenchymal cell types during development and in the adult. We have studied the importance of phosphatidylinositol-3' kinase (PI3K) signaling in these responses by mutating the PI3K-binding sites in the PDGF-beta receptor by gene targeting in embryonic stem cells. Homozygous mutant mice developed normally; however, cells derived from the mutants were less chemotactic and had largely lost their ability to contract collagen gels in response to PDGF. Injection of a mast cell degranulating agent in mice led to a decrease in interstitial fluid pressure resulting in edema formation. In contrast to wild-type mice, mutant mice were unable to normalize the pressure after treatment with PDGF. Taken together, these observations suggest a function for PDGF signaling through PI3K in interstitial fluid homeostasis by modulating the tension between cells and extracellular matrix structures.

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Figures

Figure 1
Figure 1
Introduction of point mutations at the PDGF-β receptor locus. (A) Changes introduced by in vitro mutagenesis in the amino acid sequence encompassing the PI3K-binding sites in the PDGF-β receptor are marked in bold letters. (B) Targeting of the locus. (Top) Targeting vector. (Middle) Wild-type locus. (Bottom) Targeted locus. Shaded bar, wild-type exon; solid bar, mutated exon. Oligonucleotide primers (arrowheads) used for PCR detection and probes used for Southern analysis are indicated. The position and expected fragment sizes of wild-type and targeted HindIII (H) fragments detected with the 5′ Southern are shown. E.RV, EcoRV; S, SalI; E.RI, EcoRI; H, HindIII; X, XhoI; K, KpnI; [S], site destroyed; HSV-Tk, herpes virus simplex thymidine kinase; neo-lox2, neomycin phosphotransferase expression cassette flanked by lox sites.
Figure 2
Figure 2
The mutant PDGF-β receptor is unable to associate with PI3K. Fibroblasts derived from homozygous mutant or wild-type embryos were either completely unstimulated or preincubated with PDGF-AA followed by no further stimulation or stimulation with PDGF-BB or PDGF-AA. Cell lysates derived thereof, containing equal amounts of proteins, were split in two. (A) One-half of the cell lysates was immunoprecipitated with anti-PDGF-β receptor antibodies and blotted with PY20 for the detection of kinase active β receptor. The difference in signal intensity reflects different receptor numbers of the two cell lines (data not shown). (B) The same filter was stripped and reblotted with an anti-PI3K (p85) antibody. (C) The second half of the cell lysates was immunoprecipitated with anti-PLC-γ antibody and blotted with PY20 showing tyrosine-phosphorylated PLC-γ and coimmunoprecipitated, tyrosine-phosphorylated PDGF-β receptor. (D) The same filter was stripped and reblotted with anti-PLC-γ antibody showing similar amounts of PLC-γ. ip, immunoprecipitation; ib, immunoblotting.
Figure 3
Figure 3
PI3K activity is not associated with mutant PDGF-β receptor. (A) In vitro PI3K kinase assay. TLC of PI3K reaction products from unstimulated and PDGF-BB-stimulated wild-type and homozygous mutant cells. Cells were incubated without (−) or with (+) 100 ng PDGF-BB/ml at 37°C for 5 min; cell lysates containing identical amounts of total protein (see also Fig. 2) were immunoprecipitated with β receptor-specific antiserum PDGFR3. Immunoprecipitates were subjected to PI3-kinase assay, and PI3K reaction products were analyzed by TLC and autoradiography. The positions of the reaction product, phosphatidylinositol phosphate (PIP), and the application origin are indicated. (B) Phosphorylation of PKB/Akt induced by wt or mutant PDGF receptor. Serum-starved cells were incubated in serum-free starvation medium without ligand (lanes 1 and 7) or in serum-free starvation medium with 25 ng/ml PDGF-AA (lanes 2 and 8) or 25 ng/ml PDGF-BB (lanes 3 and 9) for 5 min; to down-regulate the α receptor, cells were preincubated in serum-free starvation medium for 2 hr with 100 ng/ml PDGF-AA (lanes 4–6 and 10–12), followed by stimulation with 25 ng/ml PDGF-AA (lanes 5 and 11) or 25 ng/ml PDGF-BB (lanes 6 and 12).
Figure 4
Figure 4
PDGF-induced chemotaxis is reduced in mutant fibroblasts. Chemotactic response of wt or mutant cells toward no ligand (□) or 10 ng PDGF-BB (■) was tested by using either the “Transwell”- (A) or the “Neuroprobe”-modified Boyden Chamber assay (B; see Materials and Methods). Fold induction values are added above the bars for SD. Results from one representative experiment are shown.
Figure 5
Figure 5
PDGF-induced collagen gel contraction depends on PI3K signaling. Embryonic fibroblast-populated collagen gels were formed in serum-free MCDB 104 medium in microtiter plates. Time curves for spontaneous contraction (squares), FCS-stimulated contraction (circles), and PDGF-BB-stimulated contraction (triangles) of wt cells (open symbols) or homozygous mutant cells (filled symbols) are shown. Factors were added in triplicate in each experiment. Results from one representative experiment are shown.
Figure 6
Figure 6
Pif in the hind paw of wild-type or mutant mice after anaphylactic shock. PDGF-BB does not restore Pif after injection of PDGF-BB into mutant mice (●). SDs are given for 6 wild-type (■) animals and 11 mutant animals (●). A value of P < 0.05 was considered statistically significant.
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