Mesenchymal stromal cells (MSCs) in bone marrow are important for bone homeostasis. Although platelet-derived growth factor (PDGF) has been reported to be involved in osteogenic differentiation of MSCs, the role remains controversial and the network of PDGF signaling for MSCs has not been clarified. To clarify the underlying regulatory mechanism of MSC functions mediated by PDGF, we deleted the PDGF receptor (PDGFR)␤ gene by Cre-loxP strategy and examined the role of PDGF in osteogenic differentiation of MSCs and fracture repair. In cultured MSCs, the mRNA expression of PDGF-A, -B, -C, and -D as well as PDGFR␣ and ␤ was detected. Depletion of PDGFR␤ in MSCs decreased the mitogenic and migratory responses and enhanced osteogenic differentiation as evaluated by increased alkaline phosphatase (ALP) activity and mRNA levels of ALP, osteocalcin (OCN), bone morphogenetic protein (BMP) 2, Runx2, and osterix in quantitative RT-PCR. PDGF-BB, but not PDGF-AA, inhibited osteogenic differentiation accompanied by decreased ALP activity and mRNA levels, except for BMP2. These effects of PDGF-BB were eliminated by depletion of PDGFR␤ in MSCs except that PDGF-BB still suppressed osterix expression in PDGFR␤-depleted MSCs. Depletion of PDGFR␤ significantly increased the ratio of woven bone to callus after fracture. From the combined analyses of PDGF stimulation and specific PDGFR␤ gene deletion, we showed that PDGFR␤ signaling distinctively induces proliferative and migratory responses but strongly inhibits osteogenic differentiation of MSCs. The effects of PDGFR␣ on the osteogenic differentiation were very subtle. PDGFR␤ could represent an important target for guided tissue regeneration or tissue engineering of bone.

The neuroprotective effects of platelet-derived growth factor (PDGF) and the major signaling pathways involved in these were examined using primary cultured mouse cortical neurons subjected to H(2)O(2)-induced oxidative stress. The specific function of the PDGF beta-receptor (PDGFR-beta) was examined by the selective deletion of the corresponding gene using the Cre-loxP system in vitro. In wild-type neurons, PDGF-BB enhanced the survival of these neurons and suppressed H(2)O(2)-induced caspase-3 activation. The prosurvival effect of PDGF-AA was less than that of PDGF-BB. PDGF-BB highly activated Akt, extracellular signal-regulated kinase (ERK), c-jun amino-terminal kinase (JNK) and p38. PDGF-AA activated these molecules at lesser extent than PDGF-BB. In particular, PDGF-AA induced activation of Akt was at very low level. The neuroprotective effects of PDGF-BB were antagonized by inhibitors of phosphatidylinositol 3-kinase (PI3-K), mitogen-activated protein kinase kinase (MEK), JNK and p38. The PDGFR-beta-depleted neurons showed increased vulnerability to oxidative stress, and less responsiveness to PDGF-BB-induced cytoprotection and signal activation, in which Akt activation was most strongly suppressed. After all, these results demonstrated the neuroprotective effects of PDGF and the signaling pathways involved against oxidative stress. The effects of PDGF-BB were more potent than those of PDGF-AA. This might be due to the activation and additive effects of two PDGFRs after PDGF-BB stimulation. Furthermore, the PI3-K/Akt pathway that was deduced to be preferentially activated by PDGFR-beta may explain the potent effects of PDGF-BB.

Platelet-derived growth factors (PDGFs) and PDGF receptors (PDGFRs) are widely expressed in the mammalian CNS, though their functional significance remains unclear. The corresponding null-knockout mutations are lethal. Here, we developed novel mutant mice in which the gene encoding the b subunit of PDGFR (PDGFR-b) was genetically deleted in CNS neurons to elucidate the role of PDGFR-b, particularly in the post-natal stage. Our mutant mice reached adulthood without apparent anatomical defects. In the mutant brain, immunohistochemical analyses showed that PDGFR-b detected in neurons and in the cells in the subventricular zone of the lateral ventricle in wild-type mice was depleted, but PDGFR-b detected in blood vessels remained unaffected.The cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with controls. Furthermore, TdT-mediated dUTP-biotin nick end labeling (TUNEL)-positive neuronal cell death and lesion formation in the cerebral hemisphere were extensively exacerbated in our mutant mice after direct injection of NMDA without altered NMDA receptor expression. Our results clearly demonstrate that PDGFR-b expressed in neurons protects them from cryogenic injury and NMDA-induced excitotoxicity.

A number of receptor tyrosine kinases (RTKs) and the downstream phosphatidylinositol-3-kinase (PI3K)/Akt and mitogen-activated protein (MAP) kinase signaling pathways have been critically involved in peripheral nerve regeneration. Here, we examined the activation of PI3K/Akt and MAP kinase pathways, and platelet-derived growth factor receptors (PDGFRs) in the distal segments of crushed rat sciatic nerve from 3 to 28 days after injury. In Western blot analyses, the phosphorylated forms of extracellular signal-regulated protein kinase (ERK) and c-Jun NH(2)-terminal kinases (JNKs) were highly augmented on days 3 and 7 and on days 7 and 14 after injury, respectively. Phosphorylated Akt and p38 consistently increased from 3 to 28 days after injury. Phosphorylated PDGFR-alpha and -beta were also increased from 3 to 14 days. In the immunohistological analyses, phosphorylated ERK and PDGFR-alpha were co-localized in many activated Schwann cells and regrowing axons 3 days after injury, while PDGFR-beta was localized in a few spindle-shaped cells. The detected temporal profile of RTK signaling appears to be crucial for the regulation of Schwann cell proliferation and following redifferentiation. Furthermore, the immunohistological studies suggested a role of ERK and PDGFR-alpha in axon regeneration as well.

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