PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Arthritis Rheum. Author manuscript; available in PMC 2010 August 24.
Published in final edited form as:
PMCID: PMC2927097
NIHMSID: NIHMS151878

Differential requirements for IKKα and IKKβ in the differentiation of primary human osteoarthritic chondrocytes

Abstract

Objective

Osteoarthritic chondrocytes behave in an intrinsically de-regulated manner characterized by the chronic loss of healthy cartilage and inappropriate differentiation to a hypertrophic-like state. IKKβ and IKKα are essential kinases that activate NF-κB transcription factors, which regulate cellular differentiation and inflammation. This study reveals differential roles for each IKK in chondrocyte differentiation and hypertrophy.

Methods

Expression of IKKα or IKKβ were ablated in primary human chondrocytes by retro-transduction of specific shRNAs. Micromass cultures that faithfully undergo chondrogenesis to the terminal hypertrophic stage were established and ECM anabolism and remodelling were investigated by biochemical, immunohistochemical and ultrastructural techniques. Cellular parameters of hypertrophy (i.e. proliferation, viability and size) were also analyzed.

Results

Extracellular matrix remodelling and mineralization, processes characteristic of terminally differentiated hypertrophic cells, were defective upon IKKα or IKKβ loss. Silencing IKKβ markedly enhanced glycosaminoglycan accumulation, in conjunction with increased Sox9 expression. IKKα ablation dramatically enhanced Col2 deposition independent of Sox9 protein levels but instead in association with RUNX-2 suppression. Moreover IKKα deficient cells retained the phenotypes of pre-hypertrophic-like cells as evidenced by their smaller size and faster proliferation prior to micromass seeding along with the enhanced viability of their differentiated micromasses.

Conclusions

IKKα and IKKβ exert differential roles in ECM remodeling and endochondral ossification, events characteristic of hypertrophic chondrocytes and also factors often complicating osteoarthritis. Because IKKα's effects were more profound and pleotrophic in nature our observations suggest that exacerbated IKKα activity may be responsible for at least part of the characteristic abnormal phenotypes of osteoarthritic chondrocytes.

Keywords: NF-κB, IKKs, osteoarthritis, chondrocytes, shRNA retroviruses

Introduction

Osteoarthritis, the rheumatic disease with the highest prevalence and economic impact, is a degenerative malady driven by inflammatory factors, despite the lack of classical inflammation (1). These inflammatory factors activate chondrocytes, the unique cell component in cartilage, which are then no longer able to maintain tissue homeostasis. Chondrocytes undergo a number of cell reaction patterns including hypertrophy and terminal differentiation, as occurs in the terminal hypertrophic zone of the growth plate (2). Endochondral ossification is the final outcome of the “chondrogenic” differentiation program, which begins with chondro-progenitor proliferation from mesenchymal stem cells and ends in cartilage matrix calcification (3). RNA and protein expression patterns suggest that normal chondrocytes are kept in a state of “maturational arrest” (4). In contrast to the latter phenotype, osteoarthritic chondrocytes reveal increased expression of hypertrophic hallmarks (i.e., collagen X, alkaline phosphatase, MMP-13) suggesting that such OA derived chondrocytes have differentiated into a mature phenotype (2, 3). Because OA chondrocytes appear to be inappropriately pushed towards a hypertrophic-like state, endochondral ossification has also been proposed as a “developmental model” to understand OA pathogenesis (5). Under appropriate culture conditions, chondrocytes also exhibit a level of phenotypic plasticity comparable to mesenchymal stem cells undergoing chondrogenesis by giving rise to adipose tissue, cartilage and bone with the latter underlying a change in lineage commitment from hypertrophic chondrocytes to osteoblast-like cells (6, 7).

A variety of signaling pathways and transcription factors are known to play stage specific roles in chondrogenesis {reviewed in (3)}. TNFα mediated activation of canonical Nuclear Factor-kappaB (NF-κB) transcription factors was shown to inhibit mesenchymal chondrocytic differentiation through the post-transcriptional down-modulation of Sox9, a master chondrocytic transcription factor (8). The process of chondrogenesis is orchestrated by interactions between Sox9 and RUNX-2, which determine the fate of the differentiated chondrocyte to remain within cartilage or undergo hypertrophic maturation prior to ossification (3, 9). Sox9 has opposite effects at the early pre-hypertrophic and terminal hypertrophic phases of chondrogenesis (3, 9). Down-regulation of Sox9 (8) by NF-κBs could reflect the latter's ability to promote chondrogenic progression (3, 10). NF-κBs are well known to orchestrate most pro-inflammatory processes and thus represent a potential therapeutic target in Osteoarthritis (11). NF-κBs provide functional connections between inflammatory-like responses, normal vs. abnormal cellular growth and developmental programming (12, 13). NF-κBs act as homodimers and heterodimers of 5 different subunits that are kept in a cytoplasmic inactive state in complexes with inhibitory IκB proteins (14).

IKKα and IKKβ initiate the release of active NF-κBs from IkBs (12-14). In response to a host of pro-inflammatory stimuli, IKKβ is the dominant IκBα kinase in vivo, whose activation is essential for the nuclear translocation of canonical NF-κB heterodimers (including p65/RelA:p50 and cRel:p50). In contrast IKKα only occasionally acts as the IκBα kinase (15) but instead has been reported to activate canonical NF-κB targets in established cells (16) by acting as a nucleosomal kinase (17, 18). Unlike IKKβ, IKKα is uniquely required in vivo for the activation of the non-canonical or alternate NF-κB pathway (12). The latter involves the targeted phosphorylation and processing of the p100 precursor of the NF-κB p52 subunit, which liberates p65/p52 and RelB/p52 heterodimers to activate other NF-κB targets (12, 19). Recently IKKα was shown to activate a subset of cytoplasmic p50/p65 complexes tethered to p100 (20). Although the IKKα dependent non-canonical NF-κB pathway is not essential for normal mouse development (21, 22). IKKα has a kinase independent function that is essential for keratinocyte differentiation and can also act as a serine-threonine-kinase outside of the NF-κB pathway {reviewed in (23)}. In addition to their epidermal differentiation block, IKKα KO mice also presented selective skeletal abnormalities, which were suggestive of incomplete and/or asymmetric ossification; but the latter defects were subsequently attributed to collateral effects of systemically high FGF activities produced by their undifferentiated epidermis (24).

In the present study, we have investigated the contributions of IKKα and IKKβ in the physiology and differentiation of osteoarthritic chondrocytes. To this end IKKα and IKKβ expression were individually ablated in primary human chondrocytes by retroviral transduction of specific short hairpin (sh) RNAs. The effects of the IKK knockdowns (KDs) on the ultrastructural organization of the major ECM components (proteoglycans and Col2), and other factors indicative of chondrocyte hypertrophy (including cell size and Col10) and terminal differentiation (cell viability and calcium deposition) were evaluated in proliferating chondrocytes and their differentiated micromasses.

Materials and Methods

Isolation and preparation of primary osteoarthritic chondrocytes

Primary chondrocytes were derived from 15 OA patients undergoing knee arthroplasty, under University of Bologna Ethics Committee guidelines and with all patient identifiers removed. Chondrocytes were isolated from minced tissues by sequential enzymatic digestion as previously described (25) and then expanded in vitro at high density for up to one week prior to retroviral transduction. After KD validation, stably transduced chondrocytes were seeded into differentiating micromass cultures as previously described (25). In some cases, micromasses were embedded in OCT (Tissue-Tek, Sakura, USA), snap-frozen in liquid nitrogen (LN2) and stored at −80°C.

IKKα and IKKβ short hairpin RNAs in retroviral vectors

Knock-downs (KDs)of IKKα and IKKβ were achieved by transduction of early passage primary chondrocytes with retroviral vectors containing IKKα– or IKKβ–specific shRNA. IKKα– and IKKβ–specific oligonucleotides for each shRNA (shOligos) were subcloned into the pSuper.retro(Puro) moloney retroviral vector (26) according to the manufacturer (OligoEngine, Seattle, WA). To avoid potential off-target effects, up to three shOligos were designed containing 19-22 nt complementary to sequences in different exons of IKKα (Accession# AF012890) and IKKβ (Accession# AF080158). These were IKKα3 (19mer starting at Nt 1288), IKKα4 (22 mer starting at Nt 1474), IKKβ1 (19mer starting at Nt 457), IKKβ3 (21mer starting at Nt 2937) and IKKβ4 (23mer starting at Nt 2029). The phenotypes of chondrocytes stably transduced with IKKα or IKKβ specific shRNAs were compared with that of a negative control (GL2), compromising cells obtained from the same patient infected by a retroviral vector harboring a firefly luciferase-specific shRNA (27).

Retroviral transductions

Second-passage primary OA cohndrocytes were transduced by spinoculation with amphotyped retroviruses prepared from Phoenix A packaging cells (provided by Dr. Gary Nolan at Stanford University). Briefly viral supernatants were applied to cells by centrifugation at 1100×g at 32°C for 45 minutes with continued incubation for 5 hrs at 32°C in 5% CO2 followed by replacement with regular growth media. Seventy-two hours later shRNA expressing cells were selected for puromycin resistance (1.5 μg/ml) with 3 changes of media over 6 days.

Canonical NF-κB activity was inhibited by stable transduction with a derivative of a puromycin retroviral vector (BIP) that coexpresses an IκBα superrepressor (IκBαSR) (IBIP) (16, 28).

Toluidine Blue and Calcein staining

The GAG/proteoglycan profiles of differentiated micromasses were examined by toluidine blue staining (29); and calcified areas were assessed by calcein staining (30).

Quantitative GAG assays

The GAG content of the chondrocyte micromasses were quantified by dimethylmethylene blue (DMMB) assay essentially as described (31).

MMP-13 release assay

Release of MMP-13 from the micromass cultures under basal conditions or following stimulation with 100 units/ml interleukin-1β (IL-1β) for 72 hours was quantified by an enzyme-linked immunosorbent assay (ELISA). The specific ELISA used detects the MMP-13 proenzyme and its cleaved active form (25).

Transmission electron microscopy (TEM)

TEM analysis provided direct visual information on ECM organization, cellular viability, mitochondrial morphology and the presence of matrix vesicles and electron dense mineralization areas (32, 33). Cells were scored viable when both nuclear and cytoplasmic membranes appeared integral or non-viable with either membrane (necrosis) or nuclear (apoptosis) fragmentation.

Immunoblotting

IKK KD efficiencies and Sox9 protein levels were determined by immunoblotting with rabbit anti-human IKKα and IKKβ antibodies (Cell Signaling Technology, Beverly, MA) and affinity purified rabbit anti-Sox9 antibody (Chemicon, Temecula, CA) respectively. Immunoblots were performed on 20 μg of total cellular protein with bands visualized by chemiluminescence. Results were quantified by densitometry with Kodak 1D Image Analysis software (Kodak, New Haven, CT). For a normalization control, westerns were re-probed with an anti-tubulin antibody (Sigma).

Immunohistochemistry

Col2 IHC staining of 3 week micromasses was performed on 5 μm sections fixed with 4% PFA. After antigen unmasking (15 min at 37°C with 1 mg/ml pepsin in Tris-HCl pH 2.0), sections were incubated with anti-Col2 mouse monoclonal antibody (2 μg/ml) (MAB8887 Chemicon) and signals developed with a biotin/streptavidin amplified, alkaline phosphatase based detection system with fuchsin as substrate. Images were captured and quantified with a Nikon Eclipse 90i microscope equipped with NIS (Nikon Imaging Software) elements (Nikon Inc).

ECM neo-epitopes, which appear after the catalytic activity of MMPs on both major ECM components (DIPEN on aggrecan and Col2-¾ C on Col2) or aggrecanases (NITEGE), were revealed by IHC (34, 35) after antigen unmasking with 0.02 U/ml Chondroitinase ABC 20′ at RT Col2-¾ C neo-epitopes were detected with a C1,2C polyclonal rabbit antibody (IBEX Pharmaceuticals, Montreal, Canada) diluted 1:100. DIPEN and NITEGE neo-epitopes were detected with rabbit anti-sera kindly provided by Dr John Mort (Shriners Hospital for Children, Montreal, Canada) diluted to 0.33 μg/ml (35). Primary antibody signals were developed with the SuperSensitive Link Label IHC Detection System exploiting the “Multilink” goat antiserum (Biogenex, San Ramon, CA) and fast red substrate.

Immunohistochemical staining for Col10 was performed on sections of 1-, 2-, and 3-week micromass cultures, after antigen unmasking with 2 mg/ml hyaluronidase for 30′ at 37°C. Col10 was detected with anti-Col10 monoclonal antibody (diluted 1:1,000, IBEX Pharmaceuticals) followed by signal detection with the SuperSensitive MultiLink Label IHC Detection System with fast red substrate.

Immunohistochemical staining for RUNX-2 was performed with a goat anti-human RUNX-2/CBFA1 polyclonal antibody (0.5 μg/ml, AF2006; R&D Systems, Minneapolis, MN), developed with a “Goat Link” biotinylated rabbit anti-goat secondary antibody (Biogenex, San Ramon, CA) and detected as described above for ECM neoepitopes and Col10. In all cases hematoxylin counter staining was performed to reveal cell nuclei.

Flow cytometric analysis of chondrocytes

Sizes of retrovirally transduced cells from 7 patients were evaluated by their forward side scatter on a Vantage Flow Cytometer (Becton Dickinson, Mountain View, CA).

Cell proliferation analysis

Growth rates of GL2, IKKα3-knockdown and IKKβ1-knockdown chondrocytes were compared by pico green quantification of DNA of proliferating cells, which were initially seeded at 1,000 cells per well in quintuplicate in 96 well plates (28). To correct for differences in cell counts, values were calculated as percentage increase over the starting (day 0) values.

Cell Cycle Analysis

Cell cycle profiles of the IKKα3-knockdown, IKKβ1-knockdown and GL2 primary chondrocytes were analyzed in cells (at ~80% confluence) from up to 5 patients. The cell cycle profiles were determined by intracellular 4′,6-diamidino-2-phenylindole staining (5 μg/ml for 30 minutes at 37°C) of 4% paraformaldehyde-fixed cells, carried out essentially as described (36). Prior to DNA staining, cells were resuspended with 50 μl RNase ONE buffer (Promega, Madison, WI), heated to 65°C for 10 min, cooled on ice, treated with 22 U of RNase for 30 minutes at 37°C, centrifuged, and washed with phosphate buffered saline.

RNA expression analysis

Total cellular RNAs were prepared from one week micromasses of IKKα3-knockdown, IKKβ1-knockdown and GL2 primary chondrocytes as previously described (25). Col2a1 and RUNX-2 messenger RNA (mRNA) were quantified by real-time (RT) polymerase chain reaction (PCR) (using SYBR Green), with values normalized to the expression of GAPDH mRNA. Annealing temperatures were 56°C for GAPDH primers (NM_002046: forward 579-598 and reverse 701-683) and RUNX-2 primers (transcript variant 3 [NM_004348], forward 864-883 and reverse 968-949;, transcript variant 2 [NM_001015051]and transcript variant 1 [NM_001024630], forward 716-735 and reverse 820-801) and 58°C for Col2a1 primers (transcript variant 1 [NM_001844], forward 4247-4264 and reverse 4499-4485; transcript variant 2 [NM_033150], forward 4040-4057 and reverse 4292-4278).

Statistics

Non-parametric statistical methods were used, due to the limited size of primary patient data sets. The mean values of the groups were compared by the Wilcoxon matched pairs test. Data were analyzed using CSS statistical software (StatSoft, Tulsa, OK). All data are expressed as the mean ± SEM.

Results

Differential augmentation of the ECM-generating capacity of differentiated OA chondrocytes between IKKα- and IKKβ-knockdown cultures

To investigate the individual contributions of IKKβ and IKKα in the differentiation of primary human osteoarthritic chondrocytes, we ablated their expression by transduction with amphotyped retroviruses expressing shRNA specifically targeting each kinase. To avoid the possibility of off-target effects, up to three different shOligos targeted to different exons of IKKα and IKKβ were employed with cells from multiple patients; and all results were compared to the same patient chondrocytes stably infected with an irrelevant GL2 luciferase specific shRNA retrovirus as a negative control. In all cases >80% of P0 passage chondrocytes were converted to puromycin resistance within 6 days post infection and produced penetrant knock-downs (KDs) of either IKKα or IKKβ as assessed by western blot in multiple patients {i.e., IKKα3 (~92% KD in 10 infections); IKKα4 (~91% KD in 4 infections); IKKβ1 (~92% KD in 10 infections); IKKβ3 (>75% KD in 2 infections); IKKβ4 (~90% KD in 5 infections)} (see representative immunoblots in Figure 1A).

Figure 1
Effects of IKKα and IKKβ KDs and canonical NF-κB inhibition on GAG deposition

We first assessed the effects of either IKKβ or IKKα KD on the accumulation of modified glycosaminoglycans (GAG) in the ECM of 3 week differentiated micromass cultures (32, 37, 38). Toluidine blue staining revealed elevated GAG deposition in IKKβ and IKKα KD micromasses compared to GL2 controls with the highest GAG levels accumulating in the absence of IKKβ (i.e., IKKβ>IKKα>GL2) (Figure 1B). Importantly this qualitative result was confirmed by quantitative dimethylmethylene blue (DMMB) assays of micromasses from six patients with IKKα or IKKβ KDs (31) (see Figure 1C) (P values of 0.0044 and 0.0033 for IKKα KD and IKKβ KDs respectively).

Enforced expression of an IκBα super-repressor (IκBαSR) by retroviral transduction (IBIP vector) also resulted in increased GAG levels in either undifferentiated monolayer or differentiated micromass cultures with a stronger effect on the latter (see Figure 1D) (P value = 0.043 for monolayers and micromasses combined). To confirm that the activities of canonical NF-κBs were inhibited by IBIP infection, cells were stimulated with IL-1 and total cellular RNAs were subjected to quantitative real time RT-PCR analysis. As expected the expression of canonical NF-κB target genes including RANTES, IL-8, Mcp-1, IP-10 and MIP1α were inhibited in cells stably transduced by IBIP in comparison to a BIP empty vector control (data not shown).

Next we examined the consequences of IKKα and IKKβ KDs for micromass ECM organization by transmission electron microscopy (TEM). TEM analyses of one representative patient stably infected with IKK KD or GL2 retroviruses are presented in Figure 2A {see GL2, IKKβ KD and IKKα KD results in panels “a”, “b” and “c” respectively; bars of 250 nm}. Alternate IKKβ and IKKα shRNA retros harboring shRNAs targeted to different exons yielded similar results, thus ruling out off-target effects (see supplemental Figure 1). The relative degrees of accumulation of collagen fibrils and fibers in all micromasses were IKKα KD>IKKβ KD>>GL2 with the ECM of IKKα KD micromasses replete with highly organized collagen fibers. Careful visual inspection of more than 20 TEM fields for the presence of organized ECM (including collagen fibers, fibrils and GAG) in each of six patients revealed that ablation of IKKα had a more potent enhancing influence on overall ECM formation (IKKβ1 p value = 0.028 and IKKα3 p value = 0.018). (see graph in Figure 2A).

Figure 2
Effects of IKK KDs on ECM accumulation, Col2 protein, Col2a1 mRNA, Col2 neo-epitopes and secreted MMP13

Enhanced Col2 accumulation by IKKα or IKKβ KD is largely mediated at the post-translational level

In comparison to matched GL2 negative controls immunohistochemistry (IHC) analysis on sections of frozen micromasses revealed strong enhancement of Col2 deposition in the absence of either IKKβ or IKKα (Figure 2B, left panels and supplemental Figure 2A). To quantify the relative levels of Col2, IHC signals were captured with a Nikon Eclipse 90i microscope, analyzed with Nikon Imaging Software (NIS), and expressed as the percentage of signals in square micrometers per unit area (Figure 2B, right panels and supplemental Figure 2A). In addition, Western blots of collagen proteins extracted from micromass ECM by pepsin digestion also revealed higher levels of Col2 in the absence of IKKα or IKKβ with a rank order of IKKα KD>IKKβ KD>>GL2 micromasses (data not shown).

To begin to address how IKKβ and IKKα KDs alter accumulation of ECM components in osteoarthritic chondrocytes, we examined the levels of Col2a1 mRNA by quantitative real time (RT) PCR and also scored for the presence of Col2 neo-epitopes produced by metalloproteinases (MMPs) as a relative measure of ECM remodeling activity. Quantitative RT-PCR assays of total cellular RNAs extracted from 1 week old micromasses of multiple infected patient samples revealed similar ~2.5 fold increases of Col2a1 mRNA in cells deficient for either IKKα or IKKβ expression. Similar results were obtained with 9 X IKKα KDs and 6 X IKKβ KDs (P values of 0.007 and 0.027 respectively) (graph in Figure 2B and data not shown). To examine effects on the control of Col2 turnover, we next performed immunohistochemistry to score GL2, IKKα and IKKβ KD micromasses for the presence of Col2 ¾ neo-epitopes, which are produced by the action of specific metalloproteinases (MMP-1, MMP-8 and MMP-13). As shown in Figure 2C and supplemental Figure 2B, Col2-¾ neo-epitopes are clearly present in the ECM of GL2 micromasses but are largely absent from both IKKα and IKKβ KD micromasses. Similar results were obtained for the DIPEN and NITEGE neoepitopes of aggrecan, which appear after the action of MMPs and aggrecanases respectively (35). Taken together these findings suggest that: (a) ECM increases of Col2 in IKKα and IKKβ KD micromasses are only partially mediated by changes in Col2a1 mRNA levels and (b) Col2 remodeling is blocked in both IKKα and IKKβ KD micromasses, suggesting that each IKK strongly influences the accumulation of Col2 by post-translational mechanisms, presumably associated with the control of MMP activities.

Since MMP-13 is the major MMP responsible for Col2 remodeling at the hypertrophic phase of chondrogenesis (3, 5, 34, 39), we next explored the effects of each IKK KD on MMP-13 regulation. Basal and IL-1β induced levels of MMP13 secreted by micromass cultures were evaluated by quantitative ELISA assays of three independent patients in duplicate. Neither IKKβ nor IKKα KDs significantly affected basal MMP-13 levels, but IKKβ KD abolished IL-1β induced MMP-13 secretion (p value: 0.0277) (graph in Figure 2C). An inhibitory effect of IKKβ KD on IL-1β induced levels of MMP13 would also be consistent with MMP-13 being a target of the canonical NF-κB pathway (40). Even though there appears to be little if any effect on basal MMP-13 levels in IKKβ KD micromasses, the lack of pro-inflammatory induced MMP-13 expression by IKKβ KD micromasses might in part explain some of their increased Col2 accumulation. However we find no apparent association between MMP-13 levels and the absence of Col2-¾ neo-epitopes in IKKα KD micromasses.

Differential effects of IKKα and IKKβ loss on chondrogenic progression towards hypertrophy

To investigate if the effects of IKKα or IKKβ ablation on ECM accumulation could in part be explained by differential blockades of terminal differentiation, we looked for other signs of alterations in cell physiological parameters associated with chondrogenesis including: (1) cellular size and proliferation rate of monolayer cells prior to being seeded in micromasses, (2) the viability of differentiated micromass cultures and (3) the presence of calcium and mineralization deposits in long term 3 week micromasses. Primary micromass cultures are particularly well suited for this type of analysis, because akin to cartilaginous rudiments they recapitulate all steps of chondrogenesis from the early pre-hypertrophic to the terminal hypertrophic phase culminating in mineralization and subsequent ossification (37, 41, 42).

As shown in Figure 3A, flow cytometric forward side scatter (FSC) measurements revealed that IKKα KD chondrocytes were significantly smaller in size than their GL2 counterparts (six infected patients; p value = 0.027). These same cell size differences were also apparent by light microscopy after propidium iodide staining of cell nuclei (see Figure 3B). In contrast to these clear effects of IKKα KD, IKKβ loss had either no effect or a very modest one that was statistically insignificant (Figure 3A and data not shown). In addition IKKα KD chondrocytes also proliferated at a significantly faster rate than their GL2 or IKKβ KD counterparts (p value of 0.043 for growth curve days 3-7 in Figure 3C), which also correlated with ~2X as many IKKα KD cells in the S-G2 phases of the cell cycle (see Figures 3D).

Figure 3
Effect of IKKα KD on cell size, proliferation and cell cycle profile

IKKα KD micromasses exhibited enhanced cellular viability in comparison to their matched GL2 negative controls (see TEM analysis of 3 week micromasses of 7 infected patients in Figure 4A and 4B; p value = 0.0028). In contrast the viabilities of IKKβ KD micromasses were not significantly different from their matched GL2 samples (results with 6 infected patients in Figure 4A and other data now shown). Cells were considered viable when both the nuclear and cytoplasmic membranes appeared integral, along with the presence of euchromatin and were scored as non-viable with plasma membranes or nuclei appearing fragmented indicative of necrosis or apoptosis respectively (see representative TEM images of micromass cells in Figure 4B). However, IKKα and IKKβ KDs both had a similar physiologically beneficial effect on mitochondrial morphology in comparison to their GL2 controls. Whereas GL2 chondrocytes presented spherical, swollen and emptied mitochondria that are characteristic of terminally differentiated hypertrophic chondrocytes, the mitochondria in IKKα3 and IKKβ1 micromasses were elongated, larger, darker and also filled with septa indicative of a healthier physiological status (Figure 4C and supplemental Figure 3A). In addition, two week old micromasses were examined for the presence of Col10, a marker of the transition to chondrocyte hypertrophy (2-4). IHC analysis revealed clear Col10 staining in micromasses of GL2 control chondrocytes but no staining of IKKα-knockdown and only mild to weak staining of IKKβ-knockdown micromasses (see representative examples of staining results in cells from 1 patient in Figure 4D; see also similar results in cells fro another patient in Supplemental Figure 3B).

Figure 4
Effects of IKK KDs on cell viability and mitochondrial morphology

We then examined long-term micromasses for the accumulation of calcium deposits and other evidence of mineralization areas. Calcein staining revealed strong calcium deposition in control GL2 micromasses (30), but not in the absence of either IKKβ or IKKα (Figure 5A). The latter results agreed with the presence of mineralization vesicles (MV) and electron dense mineral areas (MA) revealed by TEM in control GL2 micromasses (37) (TEM picture in Figure 5A and supplemental Figure 4A). Analogous to these results enforced expression of IκBαSR also inhibited calcium deposition and the appearance of mineralization areas and vesicles in comparison to a BIP empty retroviral vector control (see TEM picture in Figure 5B and supplemental Figure 4B).

Figure 5
Effect of IKK KD or canonical NF-κB inhibition on OA chondrocyte terminal differentiation

IKKα and IKKβ KDs have different effects on Sox9 and RUNX-x2 expression

Next we determined the effects of each IKK on Sox9, (the major chondrogenic initiating transcription factor), and RUNX-2, (one of the essential transcription factors orchestrating terminal chondrogenic hypertrophy). Either IKKβ KD or enforced expression of IκBαSR (IBIP retroviral vector) resulted in significantly enhanced levels of Sox9 in primary proliferating chondrocytes, while loss of IKKα caused a reduction in Sox9 expression (Figures 6A and 6B). Six IKKα3 patient samples showed decreased SOX9 expression (p = 0.043), while IKKβ1 and IKKβ4 infections of 3 and 2 patients respectively increased Sox9 expression (p = 0.043). Moreover because the effects of enforced IκBαSR expression and IKKβ ablation were similar to each other, this strongly implies that IKKβ has intrinsic activity in osteoarthritic chondrocytes in the absence of an overt extracellular inflammatory/stress-like stimulus. Indeed since Sox9 is known to have opposing effects early and late in chondrogenesis (9), elevated levels of Sox9 in the IKKβ KD micromasses could enhance ECM formation, while simultaneously inhibiting endochondral ossification. However the pronounced inhibitory effect of IKKα KD on differentiation toward hypertrophy would appear to be Sox9 independent.

Figure 6
Effects of IKK KDs on Sox 9 and RUNX-2 expression

We next evaluated RUNX-2 expression by quantitative RT PCR and immunohistochemistry (Figure 6C and 6D). Whilst IKKβ KD had no significant effect on RUNX-2 levels, IKKα KD repressed RUNX-2 RNA levels in one week micromasses (p = 0.043, Fig. 6C) and RUNX-2 protein levels in three week long term micromasses (Fig. 6D and supplemental Figure 5).

Discussion

Evidence continues to accumulate that OA chondrocytes exhibit abnormal transcriptional programming in comparison to their normal articular counterparts (4, 43) such as the enhanced expression of a variety of inflammatory mediators including IL-1β, IL-8, MCP-1, NO and TNFα (which are all direct targets of NF-κB signaling) (1). Moreover, OA cartilage abnormal in vivo phenotypes and physiology are recapitulated in differentiating micromass cultures (44) which also parallel the step-wise process of chondrogenesis (2, 5). Indeed, across their maturation, micromasses established from OA chondrocytes show in progression the following in vitro expression patterns which reflect their in vivo counterparts: (1) deposition of Gag and type II collagen (as in healthy cartilage and growth plate chondroprogenitor proliferation and differentiation), (2) initiation of ECM remodeling (as in OA cartilage with enhanced degradation and growth plate hypertrophic cartilage), (3) appearance of type X collagen (as in hypertrophic cartilage observed in OA cartilage and growth plate), (4) continued ECM remodeling in conjunction with a loss in cell viability (as in advanced OA, where cell death is a dominant event and in growth plate terminal differentiation) and (5) calcium deposition and other evidence of mineralization (as in OA osteophytes and growth plate cartilage matrix calcification). This step-wise process was differentially impaired by ablation of IKKα or IKKβ, thus suggesting that IKKβ and IKKα are important positive effectors of ECM remodeling and terminal differentiation towards a hypertrophic-like state, with IKKα surprisingly having a more pronounced role than IKKβ. Nevertheless any in vitro culture system has its own inherent limitations with respect to direct comparisons to the in vivo scenario, but yet in vitro models serve as a crucial link between in vivo observations in patients and validation of disease hypotheses in in vivo animal models.

Inhibition of canonical NF-κB with an IκBα super-repressor or KD of IKKβ expression enhanced ECM formation in conjunction with a block in hypertrophy and endochondral ossification of primary OA chondrocytes in differentiating micromass cultures. Our results show that the enhanced GAG and Col2 deposition in the absence of IKKβ is contributed by a combination of factors: (a) increased anabolism caused at least in part by elevated Sox 9 levels, (b) reduced catabolism exemplified by a suppression in ECM remodeling in conjunction with a differentiation block preventing terminal calcification. Some of these observations are in keeping with and extend earlier work wherein inhibition of canonical NF-κB activation enhanced the early GAG deposition phase of chondrogenesis in murine chondrocytic cells, which was correlated with the post-transcriptional enhancement of Sox9 mRNA levels (8, 45). However our results indicate that anabolic effects are not the major cause for enhanced Col2 accumulation by IKKβ KD and even more so in IKKα KD micromasses, which are rather mostly due to a post-translational phenomenon linked to ECM remodeling suppression. Moreover we find that the canonical NF-κB pathway via IKKβ signaling takes on an intrinsic role in the context of differentiating osteoarthritic chondrocytes. Indeed our results reveal that the major consequence of IKKβ ablation (mirrored by the action of an IκBα super repressor) on chondrocyte physiology is a terminal differentiation blockade. This is in keeping with our other observations showing that pharmacological or IκBα SR mediated NF-κB inhibition in immortalized chondrocyte cell lines hampers their pro-inflammatory induced expression of known promoters (IL-8) or markers (MMP-13) of chondrocyte hypertrophy (28) and work from others showing that activators as well as downstream effectors of canonical NF-κB signaling regulate the expression of collagenase 3 (MMP-13) (46-48).

IKKα ablation had a broader range of effects on OA chondrocyte physiology. IKKα KD dramatically enhanced ECM formation as evidenced by the accumulation of highly organized Col2 fibers, a phenomenon that was independent of Sox9 levels but instead associated with RUNX-2 suppression. Increased Col2 deposition in IKKα KD micromasses was largely due to strong inhibition of collagen remodeling as evidenced by the general absence of MMP specific Col2 neo-epitopes, which was also observed by ablating IKKβ. Interestingly, this pronounced ECM remodeling block appeared to be independent of effects on basal MMP-13 levels, but possibly dependent on its activity being actively suppressed in IKK KD micromasses, an interesting possibility which is under further investigation. Moreover, IKKα ablation uniquely resulted in increased proliferative capacity and reduced cell size of undifferentiated chondrocytes and enhanced survival of differentiated micromass cultures. The enhanced viability of IKKα KD cells is not likely due to stronger, ECM dependent survival signals, because ECM was well preserved in IKKα KD micromasses, which even presented higher GAG content than IKKα KD cells. Rather, it is more likely that the selective blockage of the canonical activation pathway resulting from IKKβ- knockdown abrogates NF-κB's anti-apoptotic activity. Thus our findings suggest that the loss or inhibition of IKKα expression could potentially ameliorate the degenerative aspects of osteoarthritis that are characterized by exacerbated endochondral ossification, excessive ECM remodeling, coupled with increased cell death, each of which are complications of osteoarthritic disease. Moreover, because IKKα KD increased the replicative potential and survival of OA chondrocytes this could also provide an additional benefit for attenuating the stress induced senescence of OA chondrocytes.

OA pathogenesis is a complex phenomenon triggered by abnormal joint biomechanics and also occurring in aged cartilage, in which cellular senescence has been documented at the molecular level (49). Unlike osteoarthritic chondrocytes, proliferating chondrocytes in normal articular cartilage progress through their differentiation steps without being ”inappropriately pushed” to exit their pre-hypertrophic “ECM forming time window” to prematurely undergo terminal ECM remodeling and endochondral ossification (3, 4, 43). “Maturational arrest” of normal chondrocytes appears to be a dynamic state maintained by molecular constraints, whose failure results in their inappropriate maturation towards a hypertrophic phenotype (4). In a scale of propensity to hypertrophy, OA or aged chondrocytes are poles apart compared to normal chondrocytes, which however under particular “mineralizing” culture conditions can be conduced to progress towards hypertrophy and terminal differentiation (50). Conceivably IKKα activity could drive either OA or aged normal chondrocytes into a mature, “terminal” phenotype. Even if we find in the future that IKKα has similar properties in normal (i.e., non-OA) chondrocytes from aged matched donors under in vitro conditions, we propose that IKKα can still be considered a potential target for ameliorating the more damaging effects of OA and also for maintaining articular chondrocytes in “maturational arrest”. Moreover defining the effects of IKKα ablation for cartilage as either a template for bone formation or as a target of ectopic endochondral ossification (as occurs in OA) now represent important goals of future in vivo experiments in the context of an appropriate animal model. In this regard, we suggest that with the exception of articular cartilage, since the final outcome of terminal chondrocyte differentiation is normal bone formation, the loss of IKKα in vivo would conceivably be found to be of less clinical relevance for old patients with debilitating OA disease.

In conclusion our observations surprisingly reveal that exacerbated IKKα activity may be responsible for at least part of the idiosyncratic behavior of osteoarthritic chondrocytes including their accentuated collagen remodeling and ectopic ossification in vivo often associated with the appearance of osteophytes. Future work will be devoted to defining the mechanisms of action and downstream targets of IKKα involved in the chondrogenic programming of osteoarthritic cells and their normal counterparts.

Supplementary Material

Supplementary Figures and Legends

Acknowledgements

This work was supported by the Rizzoli Institute, the Carisbo Foundation (EO, RV and SP), a MIUR 40% Grant (FF, EF and AF), a Rientro dei Cervelli award (AF and KBM) and the MAIN EU FPVI Network of Excellence (OE, MP and KBM). KBM was a senior scholar of the Institute of Advanced Studies of the University of Bologna during most of this work.

Footnotes

The authors declare no conflicting financial interests.

References

1. Attur MG, Dave M, Akamatsu M, Katoh M, Amin AR. Osteoarthritis or osteoarthrosis: the definition of inflammation becomes a semantic issue in the genomic era of molecular medicine. Osteoarthritis Cartilage. 2002;10(1):1–4. [PubMed]
2. Sandell LJ, Aigner T. Articular cartilage and changes in arthritis. An introduction: cell biology of osteoarthritis. Arthritis Res. 2001;3(2):107–13. [PMC free article] [PubMed]
3. Goldring MB, Tsuchimochi K, Ijiri K. The control of chondrogenesis. J Cell Biochem. 2006;97(1):33–44. [PubMed]
4. Drissi H, Zuscik M, Rosier R, O'Keefe R. Transcriptional regulation of chondrocyte maturation: potential involvement of transcription factors in OA pathogenesis. Mol Aspects Med. 2005;26(3):169–79. [PubMed]
5. Aigner T, Bartnik E, Sohler F, Zimmer R. Functional genomics of osteoarthritis: on the way to evaluate disease hypotheses. Clin Orthop Relat Res. 2004;(427 Suppl):S138–43. [PubMed]
6. Roach HI, Erenpreisa J, Aigner T. Osteogenic differentiation of hypertrophic chondrocytes involves asymmetric cell divisions and apoptosis. J Cell Biol. 1995;131(2):483–94. [PMC free article] [PubMed]
7. Tallheden T, Dennis JE, Lennon DP, Sjogren-Jansson E, Caplan AI, Lindahl A. Phenotypic plasticity of human articular chondrocytes. J Bone Joint Surg Am. 2003;85-A(Suppl 2):93–100. [PubMed]
8. Sitcheran R, Cogswell PC, Baldwin AS., Jr NF-kappaB mediates inhibition of mesenchymal cell differentiation through a posttranscriptional gene silencing mechanism. Genes Dev. 2003;17(19):2368–73. [PubMed]
9. Ikeda T, Kawguchi H, Kamekura S, Ogata N, Mori Y, Nakamura K, et al. Distinct roles of Sox5, Sox6 and Sox9 in different stages of chondrogenic differentiation. J. Bone Miner Metab. 2005;23:337–340. [PubMed]
10. Feng JQ, Xing L, Zhang JH, Zhao M, Horn D, Chan J, et al. NF-{kappa}B Specifically Activates BMP-2 Gene Expression in Growth Plate Chondrocytes in Vivo and in a Chondrocyte Cell Line in Vitro. J. Biol. Chem. 2003;278(31):29130–29135. [PubMed]
11. Roman-Blas JA, Jimenez SA. NF-kappaB as a potential therapeutic target in osteoarthritis and rheumatoid arthritis. Osteoarthritis Cartilage. 2006;14(9):839–48. [PubMed]
12. Bonizzi G, Karin M. The two NF-kappaB activation pathways and their role in innate and adaptive immunity. Trends Immunol. 2004;25(6):280–8. [PubMed]
13. Karin M, Greten FR. NF-kappaB: linking inflammation and immunity to cancer development and progression. Nat Rev Immunol. 2005;5(10):749–59. [PubMed]
14. Hoffmann A, Natoli G, Ghosh G. Transcriptional regulation via the NF-kappaB signaling module. Oncogene. 2006;25(51):6706–16. [PubMed]
15. Hansberger MW, Campbell JA, Danthi P, Arrate P, Pennington KN, Marcu KB, et al. IkappaB kinase subunits alpha and gamma are required for activation of NF-kappaB and induction of apoptosis by mammalian reovirus. J Virol. 2007;81(3):1360–71. Epub 2006 Nov 22. [PMC free article] [PubMed]
16. Li X, Massa PE, Hanidu A, Peet GW, Aro P, Savitt A, et al. IKKalpha , IKKbeta , and NEMO/IKKgamma are each required for the NF-kappa B-mediated Inflammatory response program. J. Biol. Chem. 2002;277(47):45129–45140. [PMC free article] [PubMed]
17. Anest V, Hanson JL, Cogswell PC, Steinbrecher KA, Strahl BD, Baldwin AS. A nucleosomal function for IkappaB kinase-alpha in NF-kappaB-dependent gene expression. Nature. 2003;423(6940):659–63. [PubMed]
18. Yamamoto Y, Verma UN, Prajapati S, Kwak YT, Gaynor RB. Histone H3 phosphorylation by IKK-alpha is critical for cytokine-induced gene expression. Nature. 2003;423(6940):655–9. [PubMed]
19. Yang CH, Murti A, Pfeffer LM. Interferon Induces NF-{kappa}B-inducing Kinase/Tumor Necrosis Factor Receptor-associated Factor-dependent NF-{kappa}B Activation to Promote Cell Survival. J. Biol. Chem. 2005;280(36):31530–31536. [PMC free article] [PubMed]
20. Basak S, Kim H, Kearns JD, Tergaonkar V, O'Dea B, Werner SL, et al. A Fourth I□B protein within the NF-κB Signaling Module. Cell. 2007;128:369–381. [PMC free article] [PubMed]
21. Cao Y, Bonizzi G, Seagroves TN, Greten FR, Johnson R, Schmidt EV, et al. IKKalpha Provides an Essential Link between RANK Signaling and Cyclin D1 Expression during Mammary Gland Development. Cell. 2001;107(6):763–75. [PubMed]
22. Caamano JH, Rizzo CA, Durham SK, Barton DS, Raventos-Suarez C, Snapper CM, et al. Nuclear factor (NF)-kappa B2 (p100/p52) is required for normal splenic microarchitecture and B cell-mediated immune responses. J Exp Med. 1998;187(2):185–96. [PMC free article] [PubMed]
23. Perkins ND. Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat Rev Mol Cell Biol. 2007;8(1):49–62. [PubMed]
24. Sil AK, Maeda S, Sano Y, Roop DR, Karin M. IkappaB kinase-alpha acts in the epidermis to control skeletal and craniofacial morphogenesis. Nature. 2004;428(6983):660–4. [PubMed]
25. Olivotto E, Vitellozzi R, Fernandez P, Falcieri E, Battistelli M, Burattini S, et al. Chondrocyte hypertrophy and apoptosis induced by GROalpha require three-dimensional interaction with the extracellular matrix and a co-receptor role of chondroitin sulfate and are associated with the mitochondrial splicing variant of cathepsin B. J Cell Physiol. 2007;210(2):417–27. [PubMed]
26. Brummelkamp T, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell. 2002;2(3):243. [PubMed]
27. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001;411(6836):494–8. [PubMed]
28. Facchini A, Borzi RM, Marcu KB, Stefanelli C, Olivotto E, Goldring MB, et al. Polyamine depletion inhibits NF-kappaB binding to DNA and interleukin-8 production in human chondrocytes stimulated by tumor necrosis factor-alpha. J Cell Physiol. 2005;204(3):956–63. [PMC free article] [PubMed]
29. Hyllested JL, Veje K, Ostergaard K. Histochemical studies of the extracellular matrix of human articular cartilage--a review. Osteoarthritis Cartilage. 2002;10(5):333–43. [PubMed]
30. Hale LV, Ma YF, Santerre RF. Semi-quantitative fluorescence analysis of calcein binding as a measurement of in vitro mineralization. Calcif Tissue Int. 2000;67(1):80–4. [PubMed]
31. Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta. 1986;883(2):173–7. [PubMed]
32. Battistelli M, Borzi RM, Olivotto E, Vitellozzi R, Burattini S, Facchini A, et al. Cell and matrix morpho-functional analysis in chondrocyte micromasses. Microsc Res Tech. 2005;67(6):286–95. [PubMed]
33. Kirsch T, Swoboda B, Nah H. Activation of annexin II and V expression, terminal differentiation, mineralization and apoptosis in human osteoarthritic cartilage. Osteoarthritis Cartilage. 2000;8(4):294–302. [PubMed]
34. Billinghurst RC, Dahlberg L, Ionescu M, Reiner A, Bourne R, Rorabeck C, et al. Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage. J Clin Invest. 1997;99(7):1534–45. [PMC free article] [PubMed]
35. Sztrolovics R, Alini M, Roughley PJ, Mort JS. Aggrecan degradation in human intervertebral disc and articular cartilage. Biochem J. 1997;326(Pt 1):235–41. [PubMed]
36. Mazzetti I, Magagnoli G, Paoletti S, Uguccioni M, Olivotto E, Vitellozzi R, et al. A role for chemokines in the induction of chondrocyte phenotype modulation. Arthritis Rheum. 2004;50(1):112–22. [PubMed]
37. Garimella R, Bi X, Camacho N, Sipe JB, Anderson HC. Primary culture of rat growth plate chondrocytes: an in vitro model of growth plate histotype, matrix vesicle biogenesis and mineralization. Bone. 2004;34(6):961–70. [PubMed]
38. Zhang Z, McCaffery JM, Spencer RG, Francomano CA. Hyaline cartilage engineered by chondrocytes in pellet culture: histological, immunohistochemical and ultrastructural analysis in comparison with cartilage explants. J Anat. 2004;205(3):229–37. [PubMed]
39. Ortega N, Behonick DJ, Werb Z. Matrix remodeling during endochondral ossification. Trends Cell Biol. 2004;14(2):86–93. [PMC free article] [PubMed]
40. Vincenti MP, Brinckerhoff CE. Transcriptional regulation of collagenase (MMP-1, MMP-13) genes in arthritis: integration of complex signaling pathways for the recruitment of gene-specific transcription factors. Arthritis Res. 2002;4(3):157–64. [PMC free article] [PubMed]
41. Gerstenfeld LC, Landis WJ. Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system. J Cell Biol. 1991;112(3):501–13. [PMC free article] [PubMed]
42. Chen Q, Johnson DM, Haudenschild DR, Goetinck PF. Progression and recapitulation of the chondrocyte differentiation program: cartilage matrix protein is a marker for cartilage maturation. Dev Biol. 1995;172(1):293–306. [PubMed]
43. Tew SR, Li Y, Pothacharoen P, Tweats LM, Hawkins RE, Hardingham TE. Retroviral transduction with SOX9 enhances re-expression of the chondrocyte phenotype in passaged osteoarthritic human articular chondrocytes. Osteoarthritis Cartilage. 2005;13(1):80–9. [PubMed]
44. Yang KG, Saris DB, Geuze RE, Helm YJ, Rijen MH, Verbout AJ, et al. Impact of expansion and redifferentiation conditions on chondrogenic capacity of cultured chondrocytes. Tissue Eng. 2006;12(9):2435–47. [PubMed]
45. Murakami S, Lefebvre V, de Crombrugghe B. Potent inhibition of the master chondrogenic factor Sox9 gene by interleukin-1 and tumor necrosis factor-alpha. J Biol Chem. 2000;275(5):3687–92. [PubMed]
46. Mengshol JA, Vincenti MP, Coon CI, Barchowsky A, Brinckerhoff CE. Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear factor kappaB: differential regulation of collagenase 1 and collagenase 3. Arthritis Rheum. 2000;43(4):801–11. [PubMed]
47. Liacini A, Sylvester J, Li WQ, Huang W, Dehnade F, Ahmad M, et al. Induction of matrix metalloproteinase-13 gene expression by TNF-alpha is mediated by MAP kinases, AP-1, and NF-kappaB transcription factors in articular chondrocytes. Exp Cell Res. 2003;288(1):208–17. [PubMed]
48. Ijiri K, Zerbini LF, Peng H, Correa RG, Lu B, Walsh N, et al. A novel role for GADD45beta as a mediator of MMP-13 gene expression during chondrocyte terminal differentiation. J Biol Chem. 2005;280(46):38544–55. [PMC free article] [PubMed]
49. Henrotin Y, Kurz B, Aigner T. Oxygen and reactive oxygen species in cartilage degradation: friends or foes? Osteoarthritis and Cartilage. 2005;13(8):643–654. [PubMed]
50. Oyajobi BO, Frazer A, Hollander AP, Graveley RM, Xu C, Houghton A, et al. Expression of type X collagen and matrix calcification in three-dimensional cultures of immortalized temperature-sensitive chondrocytes derived from adult human articular cartilage. J Bone Miner Res. 1998;13:432–442. [PubMed]