COX-2 Involves in the Regulation of Akt/FOXO3a/p27Kip1

Signal transduction in Human Osteoblasts

1,2Li, CJ; 2,3Chang, JK; 2,3Wang, GJ; ＋1,2Ho, ML

＋Department of Physiology1, Orthopaedic Research Center2, Department of Orthopaedics3, Kaohsiung Medical University, TAIWAN

homelin@kmu.edu.tw

INTRODUCTION

Cycloxygenase (COX), including two isoenzymes COX-1 and COX-2, is

central enzyme in catalyzing the conversion of arachidonic acid to

prostaglandins (PGs). COX-1 is known as a constitutive expressed enzyme,

while COX-2 is reported to be inducible by tissue injury or inflammation1.

However, in some organs including the central nervous system, kidneys

and the gonads, COX-2 expresses in a constitutive manner1,2. However, the

physiological role of constitutive expressed COX-2 in different tissues has

not been well understood. Osteoblast is an important cell involving in bone

formation. Osteoblasts generate osteoblastogenesis through proliferation,

differentiation and mineralization to synthesize bone matrix3. COX-2 was

reported to involve in the regulating osteoblast function that fracture

healing was significantly delayed in COX-2–/– mice comparing with

COX-1–/– or wild type controls4. Furthermore, COX-2 was found to

decreased PTEN activity and then increase Akt activation via

PG-independent pathway in pancreatic cancer cells5. Our previous study

also found that the COX-2 selective inhibitor, celecoxib significantly

suppressed human osteoblast (hOB) proliferation and this effect may relate

to the decrease of Akt phosphorylation and increase of FOXO-3a

independently from PG deficiency6. In this view, we hypothesized that

COX-2 may contribute to regulating Akt/FOXO3a/p27Kip1 signaling. In

this study, we investigated the role of COX-2 in Akt/FOXO-3a/p27Kip1

signaling in hOBs.

METHODS

The effects of COX-1 or COX-2 silencing on phosphorylated Akt and

PTEN, protein level of PTEN, FOXO3a and p27Kip1 were further tested in

this study. We also use COX-1 or COX-2 specific inhibitor as a

comparison with COX-1 or COX-2 siRNA, respectively.

RESULTS

Our results showed that COX-2 silencing significantly increased p27Kip1

mRNA expression and protein level (Fig. 1 A&B), while COX-2 selective

inhibitor, NS-398, also had a similar enhancement effect on p27Kip1 mRNA

expression in hOBs (Fig. 1C). We further found that PGE-2 replenishment

failed to reverse COX-2 silencing up-regulated p27Kip1 protein level (Fig.

1D). Moreover, COX-2 silencing significantly decreased Akt and GSK-3

phosphorylation and increased FOXO3a (Fig. 2). However, neither COX-1

silencing nor COX-1 inhibitor, valeryl salcytate, can affect p27Kip1

expression (Fig. 3 A-C). The Akt phosphorylation and FOXO3a protein

level were not changed in COX-1 silenced hOBs (Fig. 3D). Most

importantly, COX-2 silencing significantly decreased Ser380 site

phosphorylation of PTEN (inactive form) (Fig. 4A) and then increased

PTEN activity in hOBs (Fig. 4B).

Figure 1 COX-2 silencing significantly increased p27Kip1 expression as

well as COX-1 specific inhibitor, and this effect was independently from

PG deficiency.

Figure 2 Decreases of p-Akt and p-GSK-3b, and increases of FOXO3a

and p27Kip1 was found in COX-2 silenced hOBs.

Figure 3 Neither COX-1 siRNA nor inhibitor, valeryl salcytate, can affect

p27Kip1 expression.

Figure 4 COX-2 silencing inhibited PTEN phosphorylation and increased

its activity in hOBs.

DISCUSSION

This study represents the first to demonstrate COX-2 involved in

Akt/FOXO-3a/p27Kip1 signaling in hOBs. We found that COX-2 silencing

significantly enhanced PTEN activity, suppressed Akt phosphorylation,

and increased FOXO3a and p27Kip1 in hOBs. Furthermore, COX-1

silencing had no effect on Akt signaling and p27Kip1 expression. According

to our results, COX-2, but not COX-1, may involve in regulating

Akt/FOXO3a/p27kip1 signaling and proliferation in hOBs. Furthermore,

PTEN activity and phosphorylation contributes to the COX-2 regulated

Akt/FOXO3a/p27Kip1 signaling. The detail molecular mechanism that

COX-2 suppresses PTEN activity needs to be further investigation.

REFERENCES

1. Newton R, et al. J Biol Chem 1998; 273:32312-21.

2. Tsatsanis C, et al. Int J Biochem Cell Biol 2006; 38:1654-61.

3. Christenson RH. Clin Biochem 1997; 30:573-93.

4. Zhang X, et al. E. J Clin Invest 2002; 109(11): 1405-15

5. Covey TM, et al. Oncogene 2007;26:5784-92.

6. Li CJ, et al. 2009 ORS Poster

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Poster No. 703 • 56th Annual Meeting of the Orthopaedic Research Society