Juliane Heilig

Effect of different running modes on the morphological, biochemical, and mechanical properties of articular cartilage.

Mechanical loading plays an important role not solely in cartilage development, but also in cartilage degeneration. Its adaptation behavior to mechanical loading has not been clearly delineated. The aim of the study was to examine the effect of different running modes (with different muscle contraction types) on morphological, biochemical, and mechanical properties of articular cartilage in the knee of growing rats. Thirty‐six female Sprague–Dawley rats were randomly assigned into a nonactive age‐matched control (AMC), level (LEVEL), and 20° downhill (DOWN) running group (n = 12 each). Running groups were trained on a treadmill for 30 min/day, 5 days/week for 6 weeks. Immunohistochemical staining and analysis of expression for collagen II, collagen IX, cartilage oligomeric matrix protein (COMP), and matrilin‐3, histomorphometry of femoral cartilage height and femoral COMP staining height, and indentation testing of tibial articular cartilage were performed. Rats subjected to downhill running showed a significantly (P = 0.015) higher COMP staining height and a tendentially (P = 0.084) higher cartilage height in the high‐weight bearing area of femoral articular cartilage. Cartilage thickness, mechanical properties, and expression of cartilage network proteins in tibial cartilage remained unaffected by different running modes. Our data suggest that joint loading induced by eccentric muscle contractions during downhill running may lead to a site‐specific adaptation.

Neutral sphingomyelinase (SMPD3) deficiency disrupts the Golgi secretory pathway and causes growth inhibition.

Systemic loss of neutral sphingomyelinase (SMPD3) in mice leads to a novel form of systemic, juvenile hypoplasia (dwarfism). SMPD3 deficiency in mainly two growth regulating cell types contributes to the phenotype, in chondrocytes of skeletal growth zones to skeletal malformation and chondrodysplasia, and in hypothalamic neurosecretory neurons to systemic hypothalamus–pituitary–somatotropic hypoplasia. The unbiased smpd3−/− mouse mutant and derived smpd3−/− primary chondrocytes were instrumental in defining the enigmatic role underlying the systemic and cell autonomous role of SMPD3 in the Golgi compartment. Here we describe the unprecedented role of SMPD3. SMPD3 deficiency disrupts homeostasis of sphingomyelin (SM), ceramide (Cer) and diacylglycerol (DAG) in the Golgi SMPD3-SMS1 (SM-synthase1) cycle. Cer and DAG, two fusogenic intermediates, modify the membrane lipid bilayer for the initiation of vesicle formation and transport. Dysproteostasis, unfolded protein response, endoplasmic reticulum stress and apoptosis perturb the Golgi secretory pathway in the smpd3−/− mouse. Secretion of extracellular matrix proteins is arrested in chondrocytes and causes skeletal malformation and chondrodysplasia. Similarly, retarded secretion of proteo-hormones in hypothalamic neurosecretory neurons leads to hypothalamus induced combined pituitary hormone deficiency. SMPD3 in the regulation of the protein vesicular secretory pathway may become a diagnostic target in the etiology of unknown forms of juvenile growth and developmental inhibition.

Insulin-like growth factor 1 receptor (IGF1R) signaling regulates osterix expression and cartilage matrix mineralization during endochondral ossification

Abstract Insulin-like growth factor 1 receptor (IGF1R) signaling is important for bone formation via endochondral ossification. Igf1r deficient mice show proportional dwarfism and alterations in chondrocyte proliferation, hypertrophy and apoptosis within the growth plate. In addition, gene ablation in mouse demonstrated that IGF1R signaling is important for osteoblast mediated bone mineralization. However, the mineralization in the terminal hypertrophic zone of the growth plate is also an essential step in endochondral ossification preceding bone formation. Therefore, we analyzed the influence of IGF1R signaling on this process by using mice with a specific deletion of Igf1r in chondrocytes. Studies in embryonic metatarsal explant cultures showed that mineralization of the terminal hypertrophic zone was strongly reduced when IGF1R signaling was lacking. This decreased mineralization may in part result from the delay in hypertrophic differentiation in the Igf1r deficient metatarsals. However, mineralization was impaired even stronger than hypertrophy, suggesting a mineralization promoting effect of IGF signaling that is independent of hypertrophic differentiation. We found a markedly decreased osterix expression suggesting that osterix is a downstream target of IGF1R in chondrocytes. MMP13 expression was strongly reduced in metatarsals lacking the IGF1R while alkaline phosphatase expression and activity were less affected. We conclude that endogenous IGF1R signaling is important for growth plate matrix remodeling and calcification leading to bone formation and suggest that regulation of osterix expression and its downstream target MMP13 are part of the underlying mechanism.

Moderate Cyclic Tensile Strain Alters the Assembly of Cartilage Extracellular Matrix Proteins In Vitro

Mechanical loading influences the structural and mechanical properties of articular cartilage. The cartilage matrix protein collagen II essentially determines the tensile properties of the tissue and is adapted in response to loading. The collagen II network is stabilized by the collagen II-binding cartilage oligomeric matrix protein (COMP), collagen IX, and matrilin-3. However, the effect of mechanical loading on these extracellular matrix proteins is not yet understood. Therefore, the aim of this study was to investigate if and how chondrocytes assemble the extracellular matrix proteins collagen II, COMP, collagen IX, and matrilin-3 in response to mechanical loading. Primary murine chondrocytes were applied to cyclic tensile strain (6%, 0.5 Hz, 30 min per day at three consecutive days). The localization of collagen II, COMP, collagen IX, and matrilin-3 in loaded and unloaded cells was determined by immunofluorescence staining. The messenger ribo nucleic acid (mRNA) expression levels and synthesis of the proteins were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and western blots. Immunofluorescence staining demonstrated that the pattern of collagen II distribution was altered by loading. In loaded chondrocytes, collagen II containing fibrils appeared thicker and strongly co-stained for COMP and collagen IX, whereas the collagen network from unloaded cells was more diffuse and showed minor costaining. Further, the applied load led to a higher amount of COMP in the matrix, determined by western blot analysis. Our results show that moderate cyclic tensile strain altered the assembly of the extracellular collagen network. However, changes in protein amount were only observed for COMP, but not for collagen II, collagen IX, or matrilin-3. The data suggest that the adaptation to mechanical loading is not always the result of changes in RNA and/or protein expression but might also be the result of changes in matrix assembly and structure.

Collagen II regulates chondroycte integrin expression profile and differentiation

Abstract Collagen II is the major fibril-forming collagen in cartilage. Complete absence of collagen II in mice is not compatible with life and in humans mutations in the COL2A1 gene lead to osteochondrodysplasias with diverse phenotypes. However, mechanistic studies on how chondrocytes respond to a lack of collagen II in their extracellular matrix are limited. Primary mouse chondrocytes were isolated from knee joints of newborn mice and transfected with siRNA targeting Col2α1 to suppress collagen II expression. The expression of integrin receptors and matrix proteins was investigated by RT-PCR and immunoblots. The localization of matrix components was evaluated by immunostaining. Signaling pathways and the differentiation state of chondrocytes was monitored by RT-PCR and flow cytometry. We demonstrate that in the absence of collagen II chondrocytes start to produce collagen I. Some binding partners of collagen II are partially lost from the matrix while other proteins, e.g. COMP, were still found associated with the newly formed collagen network. The lack of collagen II induced changes in the expression profile of integrins. Further, we detected alterations in the Indian hedgehog/parathyroid hormone-related protein (Ihh/PTHrP) pathway that were accompanied by changes in the differentiation state of chondrocytes. Collagen II seems not to be essential for chondrocyte survival in culture but it plays an important role in maintaining chondrocyte differentiation. We suggest that a crosstalk between extracellular matrix and cells via integrins and the Ihh/PTHrP pathway is involved in regulating the differentiation state of chondrocytes.

miR-322 stabilizes MEK1 expression to inhibit RAF/MEK/ERK pathway activation in cartilage

Cartilage originates from mesenchymal cell condensations that differentiate into chondrocytes of transient growth plate cartilage or permanent cartilage of the articular joint surface and trachea. MicroRNAs fine-tune the activation of entire signaling networks and thereby modulate complex cellular responses, but so far only limited data are available on miRNAs that regulate cartilage development. Here, we characterize a miRNA that promotes the biosynthesis of a key component in the RAF/MEK/ERK pathway in cartilage. Specifically, by transcriptome profiling we identified miR-322 to be upregulated during chondrocyte differentiation. Among the various miR-322 target genes in the RAF/MEK/ERK pathway, only Mek1 was identified as a regulated target in chondrocytes. Surprisingly, an increased concentration of miR-322 stabilizes Mek1 mRNA to raise protein levels and dampen ERK1/2 phosphorylation, while cartilage-specific inactivation of miR322 in mice linked the loss of miR-322 to decreased MEK1 levels and to increased RAF/MEK/ERK pathway activation. Such mice died perinatally due to tracheal growth restriction and respiratory failure. Hence, a single miRNA can stimulate the production of an inhibitory component of a central signaling pathway to impair cartilage development. Summary: miR-322 is identified as an unconventional inhibitor of a central signaling pathway in mouse chondrocytes that regulates transient and permanent cartilage homeostasis through MEK1 production and suppression of ERK1/2 phosphorylation

Impact of Arginine to Cysteine Mutations in Collagen II on Protein Secretion and Cell Survival

Inherited point mutations in collagen II in humans affecting mainly cartilage are broadly classified as chondrodysplasias. Most mutations occur in the glycine (Gly) of the Gly-X-Y repeats leading to destabilization of the triple helix. Arginine to cysteine substitutions that occur at either the X or Y position within the Gly-X-Y cause different phenotypes like Stickler syndrome and congenital spondyloepiphyseal dysplasia (SEDC). We investigated the consequences of arginine to cysteine substitutions (X or Y position within the Gly-X-Y) towards the N and C terminus of the triple helix. Protein expression and its secretion trafficking were analyzed. Substitutions R75C, R134C and R704C did not alter the thermal stability with respect to wild type; R740C and R789C proteins displayed significantly reduced melting temperatures (Tm) affecting thermal stability. Additionally, R740C and R789C were susceptible to proteases; in cell culture, R789C protein was further cleaved by matrix metalloproteinases (MMPs) resulting in expression of only a truncated fragment affecting its secretion and intracellular retention. Retention of misfolded R740C and R789C proteins triggered an ER stress response leading to apoptosis of the expressing cells. Arginine to cysteine mutations towards the C-terminus of the triple helix had a deleterious effect, whereas mutations towards the N-terminus of the triple helix (R75C and R134C) and R704C had less impact.

Extracellular Distribution of Collagen II and Perifibrillar Adapter Proteins in Healthy and Osteoarthritic Human Knee Joint Cartilage

Perifibrillar adapter proteins, interconnecting collagen fibrils, and linking the collagen network with the aggrecan matrix seem to play a crucial role in the pathogenesis of osteoarthritis (OA). Therefore, we examined immunohistochemically the extracellular distribution of collagen II and the main perifibrillar adapter proteins—collagen IX, decorin, cartilage oligomeric matrix protein (COMP), and matrilin-3—in human samples of healthy (n=4) and OA (n=42) knee joint cartilage. Histopathology assessment was performed using an OA score. Staining patterns were evaluated in relation to the disease stage. The perifibrillar adapter proteins were uniformly distributed in the upper zones of healthy cartilage. In moderate OA (n=8; score 14.3 ± 4.7), all proteins analyzed were locally absent in the fibrillated area or the superficial and upper mid zone. In advanced OA (n=20; score 18.9 ± 5.3), they were uniformly distributed in these zones and accumulated pericellularly. Perifibrillar adapter proteins are important for the stabilization of the collagen network in the upper zones of healthy cartilage. Their degradation might be a critical event in early OA. In advanced OA, there are indications for an increased synthesis in an attempt to regenerate the lost tissue and to protect the remaining cartilage from further destruction.

DOWNHILL RUNNING INDUCES SITE-SPECIFIC ARTICULAR CARTILAGE ALTERATIONS

The mechanical performance of articular cartilage and its optimization for load-bearing function is determined by the biochemical assembly of the extracellular matrix (ECM) [Mow, 1992]. Both magnitude and type of mechanical loading have the potential to alter ECM protein synthesis of collagen II and cartilage oligomeric matrix protein (COMP), two major network stabilizing proteins, in a sitespecific manner [Kiviranta, 1988, Skioldebrand, 2010]. Downhill running as a natural form of eccentric exercise [Butterfield, 2005] has been shown to induce higher knee joint loadings compared to level running [Kuster, 1995]. However, its influence on articular cartilage assembly and mechanical properties has not yet been studied. In the present study we examined the effect of different running modes (downhill vs. level) on site-specific cartilage adaptation.