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. 2014 Mar 11;9(3):e91421.
doi: 10.1371/journal.pone.0091421. eCollection 2014.

Constitutive activation of IKK2/NF-κB impairs osteogenesis and skeletal development

Gaurav Swarnkar  1 , Kaihua Zhang  1 , Gabriel Mbalaviele  2 , Fanxin Long  1 , Yousef Abu-Amer  1
Affiliations

Constitutive activation of IKK2/NF-κB impairs osteogenesis and skeletal development

Gaurav Swarnkar et al. PLoS One. .

Abstract

Pathologic conditions impair bone homeostasis. The transcription factor NF-κB regulates bone homeostasis and is central to bone pathologies. Whereas contribution of NF-κB to heightened osteoclast activity is well-documented, the mechanisms underlying NF-κB impact on chondrocytes and osteoblasts are scarce. In this study, we examined the effect of constitutively active IKK2 (IKK2ca) on chondrogenic and osteogenic differentiation. We show that retroviral IKK2ca but not GFP, IKK2WT, or the inactive IKK2 forms IKK2KM and IKK2SSAA, strongly suppressed osteogenesis and chondrogenesis, in vitro. In order to explore the effect of constitutive NF-κB activation on bone formation in vivo, we activated this pathway in a conditional fashion. Specifically, we crossed the R26StopIKK2ca mice with mice carrying the Col2-cre in order to express IKK2ca in osteoblasts and chondrocytes. Both chondrocytes and osteoblasts derived from Col2Cre/IKK2ca expressed IKK2ca. Mice were born alive yet died shortly thereafter. Histologically, newborn Col2Cre+/RosaIKK2ca heterozygotes (Cre+IKK2ca_w/f (het)) and homozygotes (Cre+IKK2ca_f/f (KI)) showed smaller skeleton, deformed vertebrate and reduced or missing digit ossification. The width of neural arches, as well as ossification in vertebral bodies of Cre+IKK2ca_w/f and Cre+IKK2ca_f/f, was reduced or diminished. H&E staining of proximal tibia from new born pups revealed that Cre+IKK2ca_f/f displayed disorganized hypertrophic zones within the smaller epiphysis. Micro-CT analysis indicated that 4-wk old Cre+IKK2ca_w/f has abnormal trabecular bone in proximal tibia compared to WT littermates. Mechanistically, ex-vivo experiments showed that expression of differentiation markers in calvarial osteoblasts derived from newborn IKK2ca knock-in mice was diminished compared to WT-derived cells. In situ hybridization studies demonstrated that the hypertrophic chondrocyte marker type-X collagen, the pre-hypertrophic chondrocyte markers Indian hedgehog and alkaline phosphatase, and the early markers Aggrecan and type-II collagen were reduced in Cre+IKK2ca_w/f and Cre+IKK2ca_f/f mice. Altogether, the in-vitro, in vivo and ex-vivo evidence suggest that IKK2ca perturbs osteoblast and chondrocyte maturation and impairs skeletal development.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Constitutive activation of NF-κB in osteoblasts, chondrocytes and stromal cells inhibits their differentiation.
(A) Calvarial osteoblasts (cOB) were transduced using retroviral constructs pMX-GFP and pMX-IKK2-ca and incubated for the time points indicated. Western blots were used to show expression of Flag-tagged IKK2 and β-actin in cOB. (B) Expression of IκB and phospho-IκB in lysates of OBs transduced with pMx-GFP or the various forms of IKK2 as indicated. (C–D) Calvarial OBs (C), ST2 and ATDC5 cells (D) were infected with GFP, IKK2WT, IKK2ca, IKK2KD, or IKK2SSAA and incubated for 21 days with 10 ug/ml of bovine insulin (media was changed and supplemented with fresh insulin every 48 hrs). Cells were then stained with either alkaline phosphatase (ALP) or Alcian blue. Note reduced staining in IKK2ca conditions. Lower left panel (C) depicts optical density quantification of upper panel. Right panel (D) represents quantification of ST2 cell and ATDC5 staining (not shown) using arbitrary units expressed as % of control. Experiments were repeated at least three times in triplicate conditions. Asterisk represents p<0.01.
Figure 2
Figure 2. Mice expressing IKK2ca exhibit abnormal skeletal development.
(A) cOB cells were harvested form newborn mice, lysed and subjected to Western blot with Flag (IKK2) and beta-actin antibodies. (B) Neurocranium of Newborn WT, Col2Cre+/IKK2ca heterozygotes (het) and homozygotes (KI). Sutures and fontanelles are widened in Col2Cre+/IKK2ca homozygotes (asterisk). Arrows (bottom) indicate the unfused and smaller supraoccipital bone in Col2Cre+/IKK2ca heterozygotes and homozygotes, respectively. Shortened snout in homozygous knock-in (large arrow) compared with hets and WT littermates. (C–D) Newborn pups were stained with Alcian blue/Alizarin red. Bone is stained red and cartilage blue. Limbs showed reduced length of scapula in newborn Col2Cre+/IKK2ca heterozygotes and homozygotes (D) Smaller skeleton and deformed vertebrate in newborn Col2Cre+/IKK2ca heterozygotes (wf) and homozygotes (ff). Reduced or missing digit ossification in Col2Cre+/IKK2ca heterozygotes and homozygotes respectively (arrows and asterisks). Reduced or diminished ossification in vertebral bodies and skull in knock-in mice is apparent (arrow heads). (E) Dorsoventral view of vertebra showing reduced width of neural arches as well as reduced or diminished degrees of ossification in vertebral bodies (arrow) of newborn Col2Cre+/IKK2ca heterozygotes and homozygotes respectively compared to WT littermates.
Figure 3
Figure 3. IKK2ca expression in vivo dampens expression of chondrogenic and osteogenic markers.
(A) Hematoxylin and eosin (H&E) staining of proximal tibia from new born pups. Col2Cre+/IKK2ca heterozygotes and homozygotes displayed disorganization of hypertrophic zones within the smaller epiphysis compared to WT littermates. In situ detection of type-I a1(Col I) type-II (Col II), type-X collagen (Col X), Alkaline phosphatase (AP), indian hedgehog (Ihh), Sox 9, and Aggrecan (Agc1) mRNAs in growth plates of femur sections prepared from new born Col2Cre+/IKK2ca homozygotes, heterozygote and control littermates. Reduced Col I, Col-II, AP positive osteoblast and Col X, Sox9 hypertrophic chondrocytes in Col2Cre+/IKK2ca heterozygote, compared to WT control is noted. (B) Higher magnification of H&E-stained sections for new born wild type and Cre+IKK2ca homozygotes. Columnar zones are indicated with arrows (C) Alkaline phosphatase staining of cOB isolated from WT and IKK2ca knock-in mice. (D) IKK2ca affects the mRNA expression of various osteoblast-differentiation associated genes. cOB were cultured in differentiation media for 9 and 12 days, followed by mRNA isolation and qPCR for various osteoblast differentiation associated gene. While, IKK2ca expression decreases the mRNA expression of ALP, Runx2 and OCN (asterisk indicates p<0.01), it increases the mRNA expression of DKK1 and SOST which negatively regulates Wnt signaling. It also increases the RANKL/OPG ratio by increasing the expression of RANKL. Asterisks * and ** represent p<0.01 and 0.05, respectively. All experiments including the histology and qPCR were repeated 3 times (n = 3).
Figure 4
Figure 4. Expression of IKK2ca impedes bone formation and reduces bone mineral density.
(A) Micro-CT analysis of four-week old Col2Cre+/IKK2ca heterozygote (Het) and WT littermate (n = 6/group). Col2Cre+/IKK2ca heterozygote has reduced trabecular bone in proximal tibia and femur compared to WT littermate. Quantitation of bone volume (BV) over total volume (TV), trabecular number (Tb.N), trabecular thickness (Tb.Th.) and trabecular separation (Tb.Sp.) is shown. (B) Bone formation rate - 4-week old mice (n = 6/group) were injected with two consecutive labels (arrows) of calcein (7 days apart) to measure bone formation rate. Asterisk represents p<0.05.
Figure 5
Figure 5. Proposed schematic model.
IKK2ca mimics persistent NF-κB activation by inflammatory signals. In this model, however, activation of the NF-κB is restricted to the mesenchymal compartment using Coll2-cre driven expression of the active IKK2 form. IKK2ca leads to exacerbated expression of NF-κB subunits (indicated here as p50/p65). According to our data, NF-κB activation inhibits expression of osteogenic markers while stimulating expression of anti-osteogenic (SOST/sclerostin, DKK1) that antagonize Wnt binding to LRP and increasing pro-osteoclastogenic factors (RANKL) that stimulate osteoclast formation. The net effect of these actions is inhibition of bone formation and increased bone resorption.
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