PI3K/AKT regulates aggrecan gene expression by modulating Sox9 expression and activity in nucleus pulposus cells of the intervertebral disc.
The goal of the investigation was to test the hypothesis that the phosphoinositide-3 kinase (PI3K)/AKT signaling pathway regulates the expression of the major extracellular matrix component of the intervertebral disc, aggrecan, in nucleus pulposus cells. Primary rat nucleus pulposus cells were treated with PI3K inhibitor to measure changes in gene and protein expression. In addition, cells were transfected with various luciferase reporter plasmids to investigate mechanisms of regulation of aggrecan gene expression. We found that treatment of nucleus pulposus cells with a PI3K inhibitor, LY294002 resulted in decreased expression of aggrecan and a reduction in deposition of sulfated glycosaminoglycans. Moreover, pharmacological suppression or co-expression of dominant negative (DN)-PI3K or DN-AKT resulted in downregulation of aggrecan promoter activity. Expression of constitutively active (CA)-PI3K significantly induced aggrecan promoter activity. We observed that PI3K maintained Sox9 gene expression and activity: inhibition of PI3K/AKT resulted in decreased Sox9 expression, lowered promoter activity, and mediated a reduction in Sox9 transcriptional activity. PI3K effects were independent of phosphorylation status of C-terminus transactivation domain (TAD) of Sox9. Finally, we noted that in nucleus pulposus cells, PI3K signaling controlled transactivation of p300 (p300-TAD activity), an important transcriptional co-activator of Sox9. Results of these studies demonstrate for the first time that PI3K/AKT signaling controls aggrecan gene expression, in part by modulating Sox9 expression and activity in cells of the nucleus pulposus.
Cheng
Chin-Chang
CC
Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Uchiyama
Yoshiyasu
Y
Hiyama
Akihiko
A
Gajghate
Sachin
S
Shapiro
Irving M
IM
Risbud
Makarand V
MV
D053674:Aggrecans / Q000235:genetics*; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D050881:E1A-Associated p300 Protein / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D006025:Glycosaminoglycans / Q000378:metabolism; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics*; D051381:Rats; D011919:Rats, Inbred Strains; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D053674:Aggrecans; D002867:Chromones; D050881:E1A-Associated p300 Protein; D004791:Enzyme Inhibitors; D006025:Glycosaminoglycans; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D055758:SOX9 Transcription Factor; C013786:A73025; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C494362:Ep300 protein, rat; C494920:Akt1 protein, rat