10/27/2013
Disclosure Zinc finger protein, ZCCHC-6 is highly expressed in Osteoarthritis cartilage and regulates the expression The authors declare no conflict of interest Nahid Akhtar , Ph.D. of Interleukin-6 in human chondrocytes Research Assistant Professor Department of Anatomy & Neurobiology, Northeast Ohio Medical University Rootstown, OH
Ahmad Arida, M.S. Research Coordinator Department of Anatomy & Neurobiology, Northeast Ohio Medical University Rootstown, OH Nahid Akhtar Research Assistant Professor Tariq M. Haqqi, M.Phil., Ph.D. Professor Department of Anatomy & Neurobiology Department of Anatomy & Neurobiology, Northeast Ohio Medical University Northeast Ohio Medical University Rootstown, Ohio Rootstown, OH
Osteoarthritis and Interleukin-6 (IL-6) mRNA stability and nucleotidyl transferases • Pre-mRNA undergoes many post-transcriptional modifications to • Higher levels of IL-6 in serum, synovial fluid and cartilage of OA patients stabilize the mRNA. Expression levels of many clinically relevant mRNAs have been reported suggesting a role of IL-6 in OA pathophysiology including cytokines are regulated by differential mRNA stability. • Increased IL-6 levels have detrimental effects on cartilage homeostasis including collagen metabolism and cartilage repair • 3’ poly(A) tail confers mRNA Cleavage and polyadenylation of pre-mRNAs stability, their export and • IL-6 is important for IL-1β induced inhibition of proteoglycan synthesis translation into protein. Poly(A) in human articular cartilage tail length is controlled by the • IL-6 up regulates the expression of MMP-1, MMP-13 and inhibits opposing actions of deadenylation COL2A1 expression in OA chondrocytes and poly(A) addition
+ • Enzymes that catalyze “tailing” are canonical nuclear poly(A) polymerase (PAP) and they belong to a super family of DNA Livshits et al., 2009; Doss et al., 2007; reviewed in Goldring,2000; polymerase β–like nucleotidyl Westcott C and Sharif M, 1996; Kapoor et al., 2011;de Lange-Brokaar et al., 2012 transferases
mRNA stability and nucleotidyl transferases ZCCHC-6 and Osteoarthritis • Recent work also identifies enzymes such as poly(U) polymerase • ZCCHC-6 (TUTase-7) is non-canonical polymerase. It contain domains capable of interacting with RNA suggesting that mechanism for (PUP) and TUTase, which synthesize cytokine regulation may extend beyond its interaction with adapters poly(U) tails and control mRNA decay
• Although, PUPs and TUTases • Nucleotidyl transferases (such as TUT-1, TUT-4) responsible for 3’ end diverge substantially in sequence, modifications has only recently been identified in cytokine regulation their functions may overlap in cancer and immune cells (Knouf et al., 2013; Jones et al., 2009)
• Mellman et al. 2008 identified • However, the role of ZCCHC-6 in cytokine regulation and OA new PUP-related enzyme (Star-PAP pathogenesis is not known activity) which adds U’s to one type Wickens and Kwak, Science, vol 319,2008. of RNA and A’s to others
• These enzymes add a poly (U) or poly(A) tail to specific mRNAs/miRNA to control degradation/stability
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Objective ZCCHC-6 is highly expressed in damaged OA cartilage
(A) 8 (C)
* 6 4
We studied (1) whether Nucleotidyl transferase mRNA 6 2 - ZCCHC-6 is expressed in OA cartilage; and (2) whether IL 0 SC DC 20X 20X it is involved in the regulation of IL-6 expression in OA (B) 3 chondrocytes *
2
6mRNA -
1 ZCCHC
0 Damaged CartilageDamaged SC DC 10X 10X 10X Control IL-6 ZCCHC-6 IL-6 and ZCCHC-6 expression in OA cartilage. (A), Gene expression of IL-6 and (B) ZCCHC-6 was determined using TaqMan assays. (C), Immunohistochemistry of IL-6 and ZCCHC-6 in damaged OA cartilage. Control section was incubated with IgG isotype. Thereafter, sections were treated with biotinylated secondary antibody and streptavidin-peroxidase conjugate, and then were developed in DAB chromogen with nickel solution. The sections were counterstained with Safranin O. SC, smooth cartilage; DC, damaged cartilage; n=3, * p<0.05
IL-1β induces ZCCHC-6 and IL-6 expression in human OA ZCCHC-6 knockdown down regulates IL-1β-induced IL-6 chondrocytes expression in OA chondrocytes
(A) (C) 4 (D) IL-6 mRNA ZCCHC-6 mRNA (A) 1.2 (B) Non-Target control siRNA + IL-1β 20000 24h 24h * 10 3 * 1
15000 * 3 * 8 * IL-6
* 2 IL-6 10000 6 0.8 8000 6mRNA 2 *
- * 6 mRNA 6 - 5000 4 0.6 6000
1 6mRNA IL 1 * - 2 4000
0 ZCCHC 0.4 * ZCCHC-6 siRNA + IL-1β
IL-1β - + 0 0 0 2000 Signal Signal intensity IL-1β - + + + + - + + + + ZCCHC 0.2 ZCCHC6 (B) 150 kDa Time (h) 0 1 2 4 6 0 1 2 4 6 0 200 0 IL-6 ZCCHC-6 siRNA - +
24h β-actin ZCCHC-6 siRNA - + Non-target siRNA + - 150 * Non-target siRNA + - IL-1β + + /ml) IL-1β - + IL-1β + +
pg 100
(A-D) Gene and protein expression levels of IL-6 and ZCCHC-6. 6 ( 6
- 50 OA chondrocytes were stimulated with IL-1β (5ng/ml) for (A) ZCCHC-6 expression after ZCCHC6 knockdown in OA chondrocytes was quantified IL indicated time. Gene expression was determined using TaqMan using TaqMan assays. (B) After 48h of transfection with Non-target control siRNA (100 0 nM) or ZCCHC-6 siRNA (100 nM), OA chondrocytes were stimulated with IL-1β for 1h IL-1β - + assays. Protein expression was determined using Western immunoblotting. Data are represented as Mean ± SE of three and culture supernatant were applied on Human inflammation antibody array (Ray different patients (n=3) each run in duplicate. * p<0.05 Biotech). Fluorescence signal intensity were analyzed using Acuity4 software. Data are represented as Mean ± SD of three different measurements.
ZCCHC-6 knockdown alters IL-6 expression in OA chondrocytes ZCCHC-6 depletion alters IL-6 mRNA poly(A) tail length and +IL-1β stability in IL-1β stimulated OA chondrocytes
(A) 350 * 2000
300 * (A) 1.2 (B)
1500
250 *
*
200 1 β
1000 β
1
1
siRNA
6 mRNA 6
-
-
- -
150 (pg/ml) 6 -
IL 0.8
NT +IL +IL
* ZCCHC6siRNA IL 100 500 * Marker * bp 50 0.6 0 0 * ZCCHC-6 siRNA - + - + - + - + - + - + Non-target siRNA + - + - + - + - + - + - 0.4 * 500
1h 3h 6h mRNAremaining 6 1h 3h 6h - 0.2 NT siRNA 400 (A) IL-1β-induced and (B) constitutive IL-6 IL ZCCHC6 siRNA 300 - IL-1β 200 (B) mRNA and protein expression in OA 0 60
1.5 chondrocytes. After 48h of transfection with 0 20 min 60 min 120 min 360 min
40 ZCCHC-6 siRNA (100 nM) or Non-target Time after actinomycin D (min) IL-6 Poly (A) tail length 1 control siRNA (100 nM),OA chondrocytes (A) IL-6 mRNA stability in OA chondrocytes transfected with ZCCHC-6 siRNA , stimulated with
were stimulated with IL-1β for 0,1,3 and 6h. 6 (pg/ml) 6
6 mRNA 6 0.5 20 -
- * IL-1β for 1h and then treated with actinomycin D for indicated time to halt the transcription. IL IL Gene expression was determined by TaqMan Data are represented as Mean ± SE of three different patients. (B) IL-6 poly (A) tail length was 0 0 * assay. IL-6 protein levels were quantified determined by PAT assay in chondrocytes after 48h of transfection with Non-target control ZCCHC-6 siRNA - + - + using ELISA. Data are represented as Mean ± siRNA (100 nM) or ZCCHC-6 siRNA (100 nM),OA chondrocytes were stimulated with IL-1β for Non-target siRNA + - + - SE of three different patients (n=3) each run 1h. NTsiRNA, non target siRNA in duplicate. * p<0.05
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ZCCHC6 expression is not regulated by NF-ᴋB ZCCHC6 expression is regulated by JNK In human chondrocytes in human chondrocytes
(A) (B) (A) (B)
3 3 3.5 *
2.5 * 2.5
actin actin
- ZCCHC-6
3 - *
actin
- β β 2 *
β 2
2.5 * 6/ - 1.5 2 1.5 * β-actin 1
1.5 6mRNA/ ZCCHC IL-1β - + + - + - + - - 1
6mRNA/ 0.5 - * SB202190 - - + + - - - - 1 0 0.5 PD98059 - - - - + + - - SP600125 ------+ + 0.5 ZCCHC-6 150 kDa ZCCHC ZCCHC 0 0 IL-1β - + + - + - + - IL-1β - + + + β-actin SB202190 - - + + - - - - MG132 - - + - IL-1β - + + + PD98059 - - - - + + - - SC514 - - - + MG132 - - + - SP600125 ------+ + SC514 - - - + Expression of ZCCHC-6 (A) Gene (B) protein in OA chondrocytes pretreated with NF-ᴋB Role of MAPK pathway in the regulation of ZCCHC-6 (A) Gene (B) protein expression inhibitor MG132 (50 µM) or SC514 (50 µM) for 2h and then stimulated with IL-1β (5ng/ml) in OA chondrocytes pretreated with MAPKs inhibitors for 2h and then stimulated for 24h. Data are represented as Mean ± SE of three different patients. * p<0.05 with IL-1β (5ng/ml) for 24h. (n=3), * p<0.05
Summary Conclusions ZCCHC-6 expression was constitutively higher in the damaged cartilage compared to smooth cartilage obtained from OA patients. Expression of IL-6 was also higher in damaged cartilage
IL-1β-stimulation of OA chondrocytes resulted in significant up regulation of both ZCCHC-6 is highly expressed in damaged human OA ZCCHC-6 and IL-6 mRNA and protein expression cartilage. These studies also identify ZCCHC6 as a Cytokine array analysis showed that a subset of cytokines including IL-6 was post-transcriptional regulator of IL-6 expression by substantially decreased by the knockdown of ZCCHC-6 in IL-1β-stimulated chondrocytes regulating poly(A)-tail length and thus influencing ZCCHC-6 knockdown significantly decreased the constitutive and IL-1β-induced IL-6 mRNA stability. These results identify ZCCHC-6 as expression of IL-6 mRNA and protein in OA chondrocytes a potential therapeutic target for OA Poly-A tail length and IL-6 mRNA stability was reduced by ZCCHC-6 knockdown in OA chondrocytes
IL-1β-induced activation of NF-kB has no role in the regulation of ZCCHC-6 expression
ZCCHC-6 expression appears to be regulated by JNK-MAPK pathway
Acknowledgement
This study was supported by National Institute of Health grants RO1 AT-003267; RO1-AT-005520 to TMH and funds from Northeast Ohio Medical University.
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