We previously reported that prostaglandin F2 alpha (PGF2alpha) and the PGF2alpha agonist fluprostenol (Flup) increased basic fibroblast growth factor (FGF-2) mRNA and protein production in osteoblastic Py1a cells. The present report extends these studies by examining the effect of PGs on FGF receptor (FGFR) mRNA expression, the subcellular localization of FGF-2 and FGFR proteins and the signaling pathway involved. Rat osteoblatic Py1a cells were grown to confluence in ham’s F-12 medium containing 5% fetal calf serum and then serum deprived for 24 h before treatment with PGF2alpha or Flup for 2 h to 24 h. Expression of FGFR mRNA was determined by northern blot analysis. Py1a cells expressed only one FGF receptor (FGFR2). In the absence of serum Py1a cells expressed a 4 kb FGFR2 mRNA transcript. Time course experiments revealed that PGF2alpha caused a 40% and Flup a 60% reduction in FGFR2 mRNA levels at 4 h and both caused an 80% reduction after 24 h. To investigate in situ localization of FGF-2 and FGFR2 protein, binding patterns of FITC labeled antibodies to FGF-2 and FGFR2 were examined by confocal laser scanning microscopy (CLSM). Py1a cells expressed low levels of cytoplasmic and nuclear localization for both FGF-2 and FGFR2 protein. Following 24 h of treatment with PGF2alpha or Flup, FGF-2 and FGFR2 proteins accumulated at nuclear membrane and were also co-localized in the nucleus of Py1a cells. CLSM studies showed that pretreatment with cycloheximide blocked nuclear labeling for FGF-2 in response to PGF2alpha. Treatment with SU5402, a specific FGFR tyrosine kinase inhibitor, did not block PG mediated nuclear accumulation of FGF-2 or FGFR2. In contrast, pretreatment with PMA, an activator of pretein kinase C (PKC) pathway prevented the nuclear accumulation of FGF-2 and FGFR2 in response to PGF2alpha. Similar results were obtained by pretreatment with the PKC inhibitor H7. Cells treated with PGs or PMA showed increased nuclear labeling for the mitogen activated protein kinase, p44/ERK2, compared to control cultures. Pretreatment with PMA blocked both PG and PMA induced ERK2 nuclear labeling. We conclude that PGs stimulate nuclear accumulation of FGF-2 and its receptor by a PKC dependent pathway and suggest a role for p44/ERK2 in this process

Prostaglandins increase FGF-2 and FGF receptor expression and nuclear accumulation in osteoblasts via MAPK kinase

SABBIETI, Maria Giovanna;MARCHETTI, Luigi;
2002

Abstract

We previously reported that prostaglandin F2 alpha (PGF2alpha) and the PGF2alpha agonist fluprostenol (Flup) increased basic fibroblast growth factor (FGF-2) mRNA and protein production in osteoblastic Py1a cells. The present report extends these studies by examining the effect of PGs on FGF receptor (FGFR) mRNA expression, the subcellular localization of FGF-2 and FGFR proteins and the signaling pathway involved. Rat osteoblatic Py1a cells were grown to confluence in ham’s F-12 medium containing 5% fetal calf serum and then serum deprived for 24 h before treatment with PGF2alpha or Flup for 2 h to 24 h. Expression of FGFR mRNA was determined by northern blot analysis. Py1a cells expressed only one FGF receptor (FGFR2). In the absence of serum Py1a cells expressed a 4 kb FGFR2 mRNA transcript. Time course experiments revealed that PGF2alpha caused a 40% and Flup a 60% reduction in FGFR2 mRNA levels at 4 h and both caused an 80% reduction after 24 h. To investigate in situ localization of FGF-2 and FGFR2 protein, binding patterns of FITC labeled antibodies to FGF-2 and FGFR2 were examined by confocal laser scanning microscopy (CLSM). Py1a cells expressed low levels of cytoplasmic and nuclear localization for both FGF-2 and FGFR2 protein. Following 24 h of treatment with PGF2alpha or Flup, FGF-2 and FGFR2 proteins accumulated at nuclear membrane and were also co-localized in the nucleus of Py1a cells. CLSM studies showed that pretreatment with cycloheximide blocked nuclear labeling for FGF-2 in response to PGF2alpha. Treatment with SU5402, a specific FGFR tyrosine kinase inhibitor, did not block PG mediated nuclear accumulation of FGF-2 or FGFR2. In contrast, pretreatment with PMA, an activator of pretein kinase C (PKC) pathway prevented the nuclear accumulation of FGF-2 and FGFR2 in response to PGF2alpha. Similar results were obtained by pretreatment with the PKC inhibitor H7. Cells treated with PGs or PMA showed increased nuclear labeling for the mitogen activated protein kinase, p44/ERK2, compared to control cultures. Pretreatment with PMA blocked both PG and PMA induced ERK2 nuclear labeling. We conclude that PGs stimulate nuclear accumulation of FGF-2 and its receptor by a PKC dependent pathway and suggest a role for p44/ERK2 in this process
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11581/242057
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