Downregulation of osteoblast markers and induction of the glial fibrillary acidic protein by oncostatin M in osteosarcoma cells require PKCdelta and STAT3.
The effects of OSM on proliferation and differentiation of osteosarcoma and nontransformed osteoblasts were analyzed. OSM downregulates osteoblast markers but induces the glial fibrillary acidic protein by the combined activation of PKCdelta and STAT3, offering new lines of therapeutic investigations.


BACKGROUND
Oncostatin M (OSM) is a multifunctional cytokine of the interleukin-6 family implicated in embryonic development, differentiation, inflammation, and regeneration of various tissues, mainly the liver, bone, and the central nervous and hematopoietic systems. One particularity of OSM relies on its growth inhibitory and pro-differentiating effects on a variety of tumor cell lines such as melanoma, providing arguments for a therapeutic application of OSM. The objective of this study was to analyze the effects of OSM on osteosarcoma cell lines proliferation and differentiation.


METHODS
Proliferation was analyzed by 3H thymidine incorporation. Differentiation was analyzed by semiquantitative RT-PCR and immunocytochemistry for various markers. Alizarin red S staining was used to evaluate bone nodule formation. Morphological changes were studied by confocal and electron microscopy. Western blotting, kinases inhibitors, and dominant negative STAT3 were used to identified the signaling pathways implicated.


RESULTS
OSM inhibits the growth of rat osteosarcoma cell lines as well as normal osteoblasts, in correlation with induction of the cyclin-dependent kinases inhibitor p21WAF1. However, OSM reduces osteoblast markers such as alkaline phosphatase, osteocalcin, and bone sialoprotein, leading to strong inhibition of mineralized nodule formation. This inhibitory effect is restricted to mature osteoblasts and differentiated osteosarcoma because OSM effectively stimulates osteoblast markers and bone nodule formation in early, but not late, bone marrow mesenchymal stem cell (BMSC) cultures. In osteosarcoma cells or BMSC, OSM induces expression of the glial fibrillary acidic protein (GFAP) as well as morphological and ultrastructural changes, for example, elongated shape and bundles of microfilaments in cell processes. Rottlerin (PKCdelta inhibitor), and to a lesser degree UO126 (MEK/ERK inhibitor), prevents the loss of osteoblastic markers by OSM, whereas dominant negative STAT3 prevents GFAP induction.


CONCLUSIONS
These results highlight the particular gene expression profile of OSM-treated osteosarcoma cells and BMSCs, suggesting either a osteocytic or a glial-like phenotype. Together with the implication of PKCdelta, ERK1/2, and STAT3, these results offer new lines of investigations for neural cell transplantation and osteosarcoma therapy.
Laboratory of Pathophysiology of Bone Resorption and Therapy of Primitive Bone Tumors, INSERM ESPRI, Nantes, France.
Céline
C
Chipoy
Martine
M
Berreur
Séverine
S
Couillaud
Gilbert
G
Pradal
François
F
Vallette
Caroline
C
Colombeix
Françoise
F
Rédini
Dominique
D
Heymann
Frédéric
F
Blanchard
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000880:Anthraquinones / Q000494:pharmacology; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology; D001842:Bone and Bones / Q000378:metabolism; D002070:Butadienes / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000378:metabolism; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005904:Glial Fibrillary Acidic Protein / Q000096:biosynthesis*; D007150:Immunohistochemistry; D007249:Inflammation; D058574:Integrin-Binding Sialoprotein; D015850:Interleukin-6 / Q000378:metabolism; D008648:Mesoderm / Q000166:cytology; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008954:Models, Biological; D009570:Nitriles / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D015675:Osteocalcin / Q000378:metabolism; D012516:Osteosarcoma / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology*; D051745:Protein Kinase C-delta; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D012038:Regeneration; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050796:STAT3 Transcription Factor; D012795:Sialoglycoproteins / Q000378:metabolism; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology; D013936:Thymidine / Q000737:chemistry; D013997:Time Factors; D015534:Trans-Activators / Q000502:physiology*; D014162:Transfection
D000880:Anthraquinones; D002070:Butadienes; C493958:Cdkn1a protein, rat; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005904:Glial Fibrillary Acidic Protein; C546873:Ibsp protein, rat; D058574:Integrin-Binding Sialoprotein; D015850:Interleukin-6; D009570:Nitriles; D012333:RNA, Messenger; D050796:STAT3 Transcription Factor; D012795:Sialoglycoproteins; C494088:Stat3 protein, rat; D015534:Trans-Activators; C113580:U 0126; D015675:Osteocalcin; C004468:Alizarin Red S; D004247:DNA; C497260:Prkcd protein, rat; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D000469:Alkaline Phosphatase; D013936:Thymidine