<i>Flnb</i>^{<i>–/–</i>}AF tissues exhibit Collagen X expression.

<p>Left: posterior, Right: anterior. (A, A′, B, B′) RNA <i>in situ</i> staining of P15 T7 IVD sagittal sections. (A, A′) <i>Flnb</i>^<i>+/+</i> and <i>Flnb</i>^{<i>–/–</i>}<i>in situ</i> sections using sense riboprobe as a negative control. (B) <i>Flnb</i>^<i>+/+</i> <i>in situ</i> section using antisense riboprobe against ColX. ColX expression is exhibited in the hypertrophic zone of the <i>Flnb</i>^<i>+/+</i> vertebral body growth plate (black arrows) and not in the IVD. (B′) <i>Flnb</i>^{<i>–/–</i>}<i>in situ</i> section using antisense riboprobe against ColX. ColX expression is exhibited in both the vertebral body growth plate hypertrophic zone (black arrows) as well as the transformed hypertrophic <i>Flnb</i>^{<i>–/–</i>}AF cells (red arrows).</p

TGFβ and BMP Dependent Cell Fate Changes Due to Loss of Filamin B Produces Disc Degeneration and Progressive Vertebral Fusions

<div><p>Spondylocarpotarsal synostosis (SCT) is an autosomal recessive disorder characterized by progressive vertebral fusions and caused by loss of function mutations in <i>Filamin B</i> (<i>FLNB)</i>. FLNB acts as a signaling scaffold by linking the actin cytoskleteon to signal transduction systems, yet the disease mechanisms for SCT remain unclear. Employing a <i>Flnb</i> knockout mouse, we found morphologic and molecular evidence that the intervertebral discs (IVDs) of <i>Flnb</i>^{<i>–/–</i>}mice undergo rapid and progressive degeneration during postnatal development as a result of abnormal cell fate changes in the IVD, particularly the annulus fibrosus (AF). In <i>Flnb</i>^{<i>–/–</i>}mice, the AF cells lose their typical fibroblast-like characteristics and acquire the molecular and phenotypic signature of hypertrophic chondrocytes. This change is characterized by hallmarks of endochondral-like ossification including alterations in collagen matrix, expression of Collagen X, increased apoptosis, and inappropriate ossification of the disc tissue. We show that conversion of the AF cells into chondrocytes is coincident with upregulated TGFβ signaling via Smad2/3 and BMP induced p38 signaling as well as sustained activation of canonical and noncanonical target genes <i>p21</i> and <i>Ctgf</i>. These findings indicate that FLNB is involved in attenuation of TGFβ/BMP signaling and influences AF cell fate. Furthermore, we demonstrate that the IVD disruptions in <i>Flnb</i>^{<i>–/–</i>}mice resemble aging degenerative discs and reveal new insights into the molecular causes of vertebral fusions and disc degeneration.</p></div

TGFβ nuclear target RNA expression increased in absence of FLNB <i>in vivo</i>.

<p>(A,B) RT-qPCR analysis results using RNA derived from P1 mouse sternums. The expression levels of the TGFβ nuclear targets <i>CTGF</i> and <i>p21</i>, are both significantly increased in <i>Flnb</i>^{<i>–/–</i>}mouse sternums, N = 6. * = p<0.05, ** = p<0.01, data are represented as mean ± SEM. (C,D) RT-qPCR analysis results using RNA derived from P15 mouse IVDs. The expression levels of the TGFβ nuclear targets, <i>CTGF</i> and <i>p21</i>, are significantly increased in <i>Flnb</i>^{<i>–/–</i>}mouse IVDs, N = 3. *** = p<0.001, **** = p<0.0001, NS = not significant, data are represented as mean ± SEM.</p

TGFβ signaling increased in absence of FLNB <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Western blot analysis of protein lysates extracted from AF tissue of P15 pups. (B-D) Western blot analysis of protein lysates extracted from cultured primary mouse chondrocytes. (B) P-Smad3 and p-ERK levels are significantly increased both with and without TGFβ-1 (5ng/mL) stimulation in mutant cells whereas total Smad3 levels are unchanged, N = 6. (C) P-Smad2 levels are significantly increased both with and without TGFβ-1 stimulation in mutant cells, N = 6. (D) P-ERK levels are significantly increased endogenously as well as after 10 minutes of TGFβ-1 stimulation whereas total ERK levels remain unchanged, N = 6.</p

TGFβ and BMP pathway components exhibit nuclear localization in <i>Flnb</i>^{<i>–/–</i>}AF.

<p>IHC analysis of P15 mouse disc tissue paraffin sections. (A, A′, E, E′) Whole IVDs shown for orientation. (B, B′, F, F′) DAPI channel only showing nuclear staining. (C, C′, G, G′) Red channel only showing protein localization. (D) P-Smad1,5,8 (red) has a cytoplasmic localization in the <i>Flnb</i>^<i>+/+</i> AF. (D′) P-Smad1,5,8 (red) exhibits a nuclear localization as it co-localizes with the nucleus (blue) In the AF of <i>Flnb</i>^{<i>–/–</i>}mice. (H) P-Smad3 (red) has a cytoplasmic localization in the <i>Flnb</i>^<i>+/+</i> AF. (H′) P-Smad3 (red) exhibits a nuclear localization as it co-localizes with the nucleus (blue) in the AF of <i>Flnb</i>^{<i>–/–</i>}mice. (I-L) Western blot analysis of fractionated protein lysates extracted from cultured primary mouse sternal chondrocytes. (I,J) p-Smad1,5,8 levels remain unchanged in the cytoplasm but are significantly higher in the nuclei of stimulated primary chondrocytes. (K,L) p-Smad3 levels are significantly upregulated in both the cytoplasm and the nucleus without stimulation. (K,L) N = 3. NS = Not Significant, * = p<0.05, ** = p<0.01, data are represented as mean ± SEM.</p

<i>Flnb</i>^{<i>–/–</i>}IVDs exhibit disruptions in AF cell morphology; show altered ECM and markers of enhanced chondrocyte differentiation.

<p>Left: posterior, Right: anterior. (A-D) H&E staining of P15 T7 IVDs. The IVD is composed of: NP = Nucleus Pulposus, AF = Annulus Fibrosus, EP = Endplate, AC = Articular Cartilage. (A-C) P15 <i>Flnb</i>^<i>+/+</i> AF cells had a fibroblast-like appearance while <i>Flnb</i>^{<i>–/–</i>}AF cells were enlarged and exhibited more hypertrophic-like qualities (arrows). (D) The <i>Flnb</i>^{<i>–/–</i>}IVD showed a disruption of the AC hypertrophic zone as well as a fissure within the NP/AF boundary (shown by *). (E-H) Polarized imaging of picrosirius red staining of P15 mouse IVD paraffin sections. (E) <i>Flnb</i>^<i>+/+</i> IVDs exhibited a distinct structure of collagen bundles forming the lamellae of the AF. (G) The <i>Flnb</i>^{<i>–/–</i>}collagenous matrix of the posterior AF lacked an organized structure. (I,J) P15 disc tissue stained with alcian blue. The posterior AF of the <i>Flnb</i>^{<i>–/–</i>}disc contained increased proteoglycan deposition compared with <i>Flnb</i>^<i>+/+</i> (black arrow). (K,L) IHC against type II collagen. Type II collagen was increased in the anterior and posterior AF of the <i>Flnb</i>^{<i>–/–</i>}IVD (black arrows). (M,N) IHC against type I pro-collagen. Pro-type I collagen protein expression was decreased throughout the AF, end-plate and nucleus pulposus of the <i>Flnb</i>^{<i>–/–</i>}IVD. (O,P) IHC against IHH in P7 T7 IVD. The <i>Flnb</i>^{<i>–/–</i>}disc contained increased IHH protein expression in the AF compared with <i>Flnb</i>^<i>+/+</i>. (Q,R) IHC against MMP13 in P15 T7 IVD. The <i>Flnb</i>^<i>+/+</i> AF exhibited some MMP13 expression in both the posterior and anterior AF regions. MMP13 protein expression was increased throughout the AF and endplate in the absence of FLNB. (S,T) TUNEL staining of P15 T7 IVD to detect apoptotic activity. <i>Flnb</i>^<i>+/+</i> discs exhibit apoptosis almost exclusively in the hypertrophic zone of the vertebral body growth plate. In <i>Flnb</i>^{<i>–/–</i>}discs, the apoptotic zone seen in <i>Flnb</i>^<i>+/+</i> discs was no longer present and there was an increase in apoptotic cells visible within the posterior and anterior AF as well as the nucleus pulposus. Blue background represents auto-fluorescence and is shown for orientation only.</p

Illustration of tissue morphology change in <i>Flnb</i>^{<i>–/–</i>}IVD.

<p>(A) <i>Flnb</i>^<i>+/+</i> IVD illustration. (B) <i>Flnb</i>^{<i>–/–</i>}IVD illustrating transition of AF to hypertrophic-like state resembling the mineralized endplate. Flanking vertebral bodies shift in position as IVD degenerates resulting in inappropriate spinal curvature. (C) IVD of 5 year old SCT patient. Positions of flanking vertebrae suggest compression of the IVD identical to those observed in <i>Flnb</i>^{<i>–/–</i>}mouse spines.</p

BMP non-canonical pathway activation is increased in the absence of FLNB <i>in vivo</i> and <i>in vitro</i>.

<p>(A-C) Western blot analysis of protein lysates extracted from cultured primary mouse chondrocytes. (A) P-Smad1,5,8 levels remain unchanged in <i>Flnb</i>^{<i>–/–</i>}when compared with <i>Flnb</i>^<i>+/+</i>, N = 6. (B) P-p38 levels are increased endogenously as well as upon BMP-2 (10ng/mL) stimulation in mutant versus <i>Flnb</i>^<i>+/+</i> chondrocytes, N = 3. (C) p-ERK levels are upregulated endogenously but are subsequently decreased in mutant chondrocytes following 30 minutes of BMP-2 stimulation. N = 4. (D) Luciferase assay measuring <i>Msx2</i> promoter activity in primary mouse chondrocytes. <i>Msx2</i> promoter activity is increased in <i>Flnb</i>^{<i>–/–</i>}chondrocytes, N = 3. * = p<0.05, ** = p<0.01, data are represented as mean ± SEM. (E) Western blot analysis of protein lysates extracted from AF tissue of P15 pups. BMP-2 and p-p38 protein expression levels are increased in <i>Flnb</i>^{<i>–/–</i>}IVDs with no change in p-Smad1/5/8 levels, N = 7.</p

Transformed hypertrophic AF cells continue to express AF marker Scleraxis in <i>Flnb</i>^{<i>–/–</i>}IVDs.

<p>(A-D) IHC analysis of Scleraxis-GFP expression in P15 mouse disc tissue paraffin sections. (A,B) <i>Flnb</i>^<i>+/+</i> IVDs at P7 and P15 show SCX expression in the AF tissues (white arrows). (A′,B′) <i>Flnb</i>^{<i>–/–</i>}IVDs at P7 and P15 exhibit continued SCX expression in the transformed hypertrophic AF cells.</p

<i>Flnb</i>^{<i>–/–</i>}vertebrae progressively fuse in the thoracic and lumbar areas of the spine.

<p>Posterior view of mouse spines stained for cartilage proteoglycans (blue) and mineralized bone (red). (A-C, A′-C′) The <i>Flnb</i>^{<i>–/–</i>}mouse spine exhibited ectopic ossifications between the neural arches of the thoracic area (arrows). (D, D′) P15 image depicts representative vertebral fusions between thoracic vertebrae in the <i>Flnb</i>^{<i>–/–</i>}mouse spine (arrows). (E, E′) At P19, vertebral fusions and ectopic ossifications have progressed into the lumbar area (arrows). (F, F′) Anterior view. Multiple discs have disappeared and mineralized to bone in the P21 <i>Flnb</i>^{<i>–/–</i>}mouse spine. N = 3 for each timepoint.</p