Elevated inflammatory gene expression in intervertebral disc tissues in mice with ADAM8 inactivated.

We found ADAM8 enzymatic activity elevated in degenerative human intervertebral disc (IVD). Here, we examined the discs in ADAM8-inactivation mice that carry a mutation preventing self-activation of the enzyme. Surprisingly, elevated gene expression for inflammatory markers (Cxcl1, IL6) was observed in injured discs of ADAM8 mutant mice, along with elevated expression of type 2 collagen gene (Col2a1), compared with wild type controls. Injured annulus fibrosus of mutant and wild type mice contained a higher proportion of large collagen fibers compared with intact discs, as documented by microscopic examination under circular polarized light. In the intact IVDs, Adam8EQ mouse AF contained lower proportion of yellow (intermediate) fiber than WT mice. This suggests that ADAM8 may regulate inflammation and collagen fiber assembly. The seemingly contradictory findings of elevated inflammatory markers in mutant mice and excessive ADAM8 activity in human degenerative discs suggest that ADAM8 may interact with other enzymatic and pro-inflammatory processes needed for tissue maintenance and repair. As a future therapeutic intervention to retard intervertebral disc degeneration, partial inhibition of ADAM8 proteolysis may be more desirable than complete inactivation of this enzyme.

Vitamin D receptor is a novel transcriptional regulator for Axin1.

BACKGROUND: Axin1 is a scaffold protein in the ß-catenin destruction complex, which, if disrupted, contributes to pathogenesis of various human diseases, including colorectal carcinogenesis and inflammatory bowel diseases (IBD). We have previously demonstrated that Salmonella infection promotes the degradation and plasma sequestration of Axin1, leading to bacterial invasiveness and inflammatory responses. Vitamin D and the vitamin D receptor (VDR) appear to be important regulators of IBD and colon cancer. Although VDR and Axin1 are all involved in intestinal inflammation, it remains unclear whether these processes are related or function independently. In the current study, we hypothesize that VDR is an important regulator for the maintenance of physiological level of Axin1. METHODS: Using the intestinal epithelial conditional VDR knockout mouse model (VDRΔIEC) and cultured cell lines, influences of VDR status on the expression of Axin1 was evaluated by Western blots and real-time PCR. Loss- and gain-of-function assays were used to investigate the regulation of VDR on Axin1 at the transcriptional and translational levels. Cells were treated with cycloheximide or actinomycin for molecular mechanistic studies. Candidate genomic VDR binding sites for Axin1 were tested by chromatin immunoprecipitation (ChIP) assay. Physical interactions among VDR, Axin1, and ß-catenin were tested by immunoprecipitation. Cellular localization of Axin1 with different VDR status was determined by fractionation and immunohistochemistry. RESULTS: We found that VDR deletion led to lower protein and mRNA levels of Axin1, whereas knockdown of Axin1 did not change the expression level of VDR protein. Immunoprecipitation data did not support physical interaction between VDR and Axin1. The VDR regulation of Axin1 was through a VDR genomic binding site for Axin1 gene on the regulatory region. Fractionation data showed that cytosolic Axin1 was significantly reduced due to VDR deletion, leaving the nuclear fraction unchanged. In ileum, Axin1 was distributed in the cytosol of apical epithelium and crypts. CONCLUSION: VDR is important for the maintenance of physiological level of Axin1. The discovery of Axin1 as a VDR target gene provides novel and fundamental insights into the interactions between the VDR and ß-catenin signaling pathways.

Biglycan Inhibits Capsaicin-Induced Substance P Release by Cultured Dorsal Root Ganglion Neurons.

OBJECTIVE: The purpose of this study was to examine the inhibitory effects of biglycan on substance P release from cultured sensory neurons in response to capsaicin. STUDY DESIGN: In vitro study of cultured primary sensory neurons from the rabbit dorsal root ganglion (DRG). We interrogated the culture system function with capsaicin. Biglycan is an important structural component of the intervertebral disc that may regulate growth factors and inflammatory mediators. We tested the hypothesis that biglycan inhibits substance P release in response to capsaicin. RESULTS: The DRG cultures were shown to contain both neurons and astrocytes by immunostaining using antibodies recognizing neuron and glial cell markers. Cultured DRG cells respond to capsaicin in a dose- and time-dependent manner (capsaicin dose ranges from 5 to 500 µmol/L; stimulation time ranges from 0 to 60 minutes). The neurons preincubated with biglycan released 27% less substance P compared with neurons without biglycan (n = 4, P = 0.036). CONCLUSION: We have established a DRG cell culture system, which contains both sensory neurons and the supporting astrocytes. Biglycan, an inhibitor of substance P release by DRG cultures, may serve as an ingredient in intradiscal injectables to reduce back pain.

Fibronectin fragments and the cleaving enzyme ADAM-8 in the degenerative human intervertebral disc.

STUDY DESIGN: The presence of fibronectin fragments (FN-fs) and the cleaving enzyme, A disintegrin and metalloproteinase domain-containing protein (ADAM)-8 were examined in human intervertebral disc (IVD) tissue in vitro. OBJECTIVE: To investigate the presence and pathophysiological concentration of FN-fs and their cleaving enzyme, ADAM-8, in the human IVD tissue. SUMMARY OF BACKGROUND DATA: The 29-kDa FN-f has been shown to result in extracellular matrix loss in rabbit IVDs. However, the concentration of this biologically active fragment in the degenerative human IVD tissue has previously not been determined. Furthermore, it is critical to identify the enzyme(s) responsible for FN cleavage in the IVD. METHODS: Human degenerative IVD tissues were removed during spinal surgery. A normal seeming young adult and an infant human cadaveric sample were obtained as controls. Soluble proteins were extracted, and analyzed by Western blotting using antibodies specific for the human FN neoepitope VRAA²7¹. A purified 29-kDa FN-f was used to allow estimation of the concentration of FN-fs in the tissues. ADAM-8, a FN-cleaving enzyme, was analyzed by Western blotting and immunostaining. RESULTS: All adult IVD tissues contain many FN-f species, but these species were absent from the infant disc tissue. Moderately degenerative discs contained the highest amount of FN-fs; the concentration was estimated to be in the nanomolar range per gram of tissue. ADAM-8, known to cleave FN resulting in the VRAA²7¹ neoepitope, was present in the human disc. ADAM-8 primarily localized in the pericellular matrix of the nucleus pulposus tissue, as determined by immunostaining. CONCLUSION: This is the first report that N-terminal FN-fs are consistently present in IVD tissues from adult subjects. The pathophysiological concentration of these fragments is estimated to be at nanomolar range per gram of IVD tissue. Furthermore, ADAM-8, known to cleave FN, is present at the pericellular matrix of disc cells.

Lactoferricin mediates anti-inflammatory and anti-catabolic effects via inhibition of IL-1 and LPS activity in the intervertebral disc.

The catabolic cytokine interleukin-1 (IL-1) and endotoxin lipopolysaccharide (LPS) are well-known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL-1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti-catabolic and anti-inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL-1 and LPS-mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL-1 and LPS-mediated proteoglycan (PG) depletion, matrix-degrading enzyme production, and enzyme activity in long-term (alginate beads) and short-term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL-1 and LPS-mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage-degrading enzymes, including MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor-induced stimulation of oxidative and inflammatory factors such as iNOS, IL-6, and toll-like receptor-2 (TLR-2) and TLR-4. Finally, the ability of LfcinB to antagonize IL-1 and LPS-mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future.

Toll-like receptor adaptor signaling molecule MyD88 on intervertebral disk homeostasis: in vitro, ex vivo studies.

MyD88 is an adapter protein that links toll-like receptors (TLRs) and Interleukin-1 receptors (IL-1Rs) with downstream signaling molecules. The MyD88 has been found to be an essential mediator in the development of osteoarthritis in articular cartilage. However, the role of the MyD88 pathway has yet to be elucidated in the intervertebral disk (IVD). Using in vitro techniques, we analyzed the effect of MyD88 pathway-specific inhibition on the potent inflammatory and catabolic mediator LPS and IL-1 in bovine and human nucleus pulposus (NP) cells by assessing matrix-degrading enzyme expression, including matrix metalloproteases (MMPs) and a disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS family). We also analyzed inhibition of MyD88 in the regulation of inducible nitric oxide synthase and TLR-2. Finally, we used an ex vivo organ culture model to assess the effects of MyD88 inhibitor (MyD88i) on catabolic factor-induced disk degeneration in mice lumbar disks. In bovine NP cells, MyD88i potently antagonizes LPS- or IL-1-mediated induction of cartilage-degrading enzyme production, including MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5. MyD88i also attenuates the LPS- or IL-1-mediated induction of iNOS and TLR-2 gene expression. Our ex vivo findings reveal inhibition of MyD88 via counteraction of IL-1-mediated proteoglycan depletion. The findings from this study demonstrate the potent anti-inflammatory and anti-catabolic effects of inhibition of MyD88 pathway inhibition on IVD homeostasis, suggesting a potential therapeutic benefit of a MyD88i in degenerative disk disease in the future.

Biological effects of the plant-derived polyphenol resveratrol in human articular cartilage and chondrosarcoma cells.

The natural phytoestrogen resveratrol (RSV) may have therapeutic potential for arthritic conditions. RSV is chondroprotective for articular cartilage in rabbit models for arthritis, but its biological effects on human articular cartilage and chondrosarcoma cells are unknown. Effects of RSV on human articular cartilage homeostasis were studied by assessing production of matrix-degrading enzymes (MMP-13, ADAMTS4, and ADAMTS5), as well as proteoglycan production and synthesis. The counteractions of RSV against catabolic factors (e.g., FGF-2 or IL-1ß) were examined by in vitro and ex vivo using monolayer, three-dimensional alginate beads and cartilage explants cultures, respectively. RSV improves cell viability of articular chondrocytes and effectively antagonizes cartilage-degrading protease production that was initiated by catabolic and/or anti-anabolic cytokines in human articular chondrocytes. RSV significantly also enhances BMP7-promoted proteoglycan synthesis as assessed by (35) S-sulfate incorporation. Protein-DNA interaction arrays suggest that RSV inhibits the activation of transcription factors involved in inflammation and cartilage catabolic signaling pathways, including direct downstream regulators of MAPK (e.g., AP-1, PEA3) and NFκB. RSV selectively compromises survival of human chondrosarcoma cells, but not primary articular chondrocytes, revealing cell-specific activity of RSV on non-tumorigenic versus tumor-derived cells. We propose that RSV exerts its chondroprotective functions, in part, by deactivating p53-induced apoptosis in human primary chondrocytes, but not human chondrosarcoma. Our findings suggest that RSV has potential as a unique biologic treatment for both prevention and treatment of cartilage degenerative diseases.

The effects of age, sex, ethnicity, and spinal level on the rate of intervertebral disc degeneration: a review of 1712 intervertebral discs.

STUDY DESIGN: A gross anatomic and magnetic resonance imaging study of intervertebral disc (IVD) degeneration in fresh cadaveric lumbar spines. OBJECTIVE: The purpose of this study was to find the rate of IVD degeneration. SUMMARY OF BACKGROUND DATA: Age, sex, race, and lumbar level are among some of the factors that play a role in IVD degeneration. The rate at which IVDs degenerate is unknown. METHODS: Complete lumbar spine segments (T11/T12 to S1) were received within 24 hours of death. The nucleus pulposus, anulus fibrosus, cartilaginous and bony endplate, and the peripheral vertebral body were assessed with magnetic resonance imaging and IVD degeneration was graded by two observers from grade 1 (nondegenerated) to grade 5 (severely degenerated) on the basis of a scale developed by Tanaka et al. The specimens were then sectioned and gross anatomic evaluation was performed according to Thompson et al. RESULTS.: A total of 433 donors and 1712 IVDs were analyzed. There were 366 whites, 47 Africans, 16 Hispanics, 4 Asian. There were 306 male and 127 female donors. The age range was 14 to 81 years, (average: 60.5 ± 11.3). For donors greater than age 40, the L5/S1 IVD degenerated at a significantly faster rate of 0.043 per year compared to 0.031, 0.034, 0.033, 0.027 for L1/L2, L2/L3, L3/L4, L4/L5, respectively. For donors younger than 40, L5/S1 IVD degenerated at a significantly faster rate of 0.141/y compared to 0.033, 0.021, 0.031, 0.050 for L1/L2, L2/L3, L3/L4, L4/L5, respectively. Multiple regression analysis revealed that sex had no significant effect on IVD degeneration whereas African ethnicity was associated with lower Thompson score at L1/L2, L2/L3, L3/L4, L4/L5 when compared with whites. CONCLUSION: The relatively early degeneration at L5-S1 in all races and lower Thompson grade in donors of African ethnicity needs further investigation. Factors such as sagittal alignment, facet joint arthritis, and genetics potentially play a role in IVD degeneration.

Characterization of TIMP-3 in human articular talar cartilage.

Tissue inhibitor of matrix metalloproteinase 3 (TIMP-3) is an inhibitor of matrix degradation; however, little else is known about the role(s) of this protein in articular cartilage. In this study we compared levels of TIMP-3 in human knee and ankle cartilages and in normal and degraded cartilages. In addition, our studies focused on the compartmentalization of TIMP-3 in human adult articular cartilage matrix, identification of its potential binding partners, and determining the effects of cytokines on its matrix compartment deposition. We extracted TIMP-3 from cartilage and found that while TIMP-3 was localized throughout the matrix, it was predominately associated with the chondrocyte. We also found that more TIMP-3 was extracted from normal compared to degraded cartilage and more in ankle than knee cartilage suggesting the potential of this inhibitor as a protective agent. Our data suggest that TIMP-3 interacts with heparan sulfate and heparan sulfate proteoglycans and to a lesser extent with heparin and chondroitin sulfate. Stimulation with Interleukin-1ß and osteogenic protein-1 decreased while tumor necrosis factor alpha and transforming growth factor beta increased TIMP-3 protein levels; however, TIMP-3 mRNA was not significantly affected by any of these treatments. These characteristics indicate the chondroprotective nature of TIMP-3 and its potential as a therapeutic agent for osteoarthritis.

Biglycan regulates the expression of EGF receptors through EGF signaling pathways in human articular chondrocytes.

Biglycan is a member of the family of small leucine-rich proteoglycans. It is an important structural component of articular cartilage and participates in the assembly of the chondrocyte extracellular matrix through formation of protein interactions with collagen type VI and large proteoglycan aggregates. Biglycan also possesses signaling properties. In articular chondrocytes, short-term activation of epidermal growth factor receptors (EGFR) with biglycan initiated mitogen-activated protein kinase and phosphatidylinositol 3-kinase (PI3K) signaling events, similar to the effect of epidermal growth factor (EGF) observed in other cell types. The extent and duration of intracellular signaling resolves biological effects initiated by EGFR stimulation, thus, establishing cell fate. In this study, we elucidate a novel regulatory mechanism of EGFR expression in human articular chondrocytes that is modulated by prolonged biglycan treatment and is in contrast to changes detected in the expression of EGFR following EGF stimulation. Treatment of chondrocytes for 24 hr with biglycan upregulated EGFR mRNA and protein expression, whereas treatment with EGF downregulated EGFR message and protein levels. Biglycan and EGF treatment protracted extracellular signal-regulated kinases (ERK1/2) and Akt phosphorylation, albeit to different extents. Mechanistic studies with mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathway-specific inhibitors revealed that biglycan and EGF distinctly modulate the expression of EGFR in chondrocytes. Biglycan promoted the coactivation of ERK1/2 and Akt, however, phosphorylated Akt induced a prolonged inhibition of ERK1/2. Consequently, total EGFR mRNA and protein expression was increased. This regulatory mechanism contrasts the modulation of EGFR expression by exogenous EGF, which strongly protracts ERK1/2 activation, therefore, inducing a decrease of EGFR message and protein levels. Thus, biglycan might impinge on the expression of total EGFR and possibly, on the cell-surface expression of the receptors. These observations suggest that biglycan might play a critical role in the regulation of chondrocyte and pericellular matrix homeostasis.

Age-related changes in the extracellular matrix of nucleus pulposus and anulus fibrosus of human intervertebral disc.

STUDY DESIGN: To characterize age-related changes in the matrix of human intervertebral disc (IVD) specimens, human specimens from the third to the eighth decade of life were collected and analyzed for collagen and proteoglycan (PG) composition. OBJECTIVE: To identify age-related changes in the concentration of matrix macromolecules (collagen and PGs, including the small leucine-rich PGs biglycan, decorin, fibromodulin, and lumican) in human anulus fibrosus (AF) and nucleus pulposus (NP). SUMMARY OF BACKGROUND DATA: IVD degeneration is associated with changes in the concentration and fragmentation of matrix molecules. Deciphering age-related matrix alterations may help us to better understand the regulatory mechanisms underlying IVD degeneration. METHODS: Forty-six whole IVDs were obtained from the thoracolumbar spines (T11-L5) of humans aged between 32 and 80 years. All specimens were classified as Thompson grade 1 or 2 according to MRI criteria. Specimens were separated into (i) outer-and (ii) inner AF, and (iii) NP. DNA, collagen, and PG contents were measured using chemical assays, whereas small nonaggregating PG levels were analyzed by comparative Western blotting. RESULTS: Total PG and collagen contents in both the AF and NP consistently decreased with aging. The concentrations of small nonaggregating PGs varied. In the outer anulus, decorin levels decreased, whereas biglycan and fibromodulin levels increased with age. In the inner anulus and nucleus, biglycan demonstrated a significant increase with aging. These changes differed in most cases from those previously reported for degenerating disc tissues. CONCLUSION: Collagen and PGs appeared to undergo specific age-related changes in the human IVD. Although the total contents of these 2 families of molecules decreased during aging, individual species of small nonaggregating PGs showed species-specific age-related changes. Interestingly, the level of biglycan rose and remained elevated in all 3 compartments of the disc with aging. The functional significance of these alterations is yet to be determined.

Prostaglandin E2 and its cognate EP receptors control human adult articular cartilage homeostasis and are linked to the pathophysiology of osteoarthritis.

OBJECTIVE: To elucidate the pathophysiologic links between prostaglandin E(2) (PGE(2)) and osteoarthritis (OA) by characterizing the catabolic effects of PGE(2) and its unique receptors in human adult articular chondrocytes. METHODS: Human adult articular chondrocytes were cultured in monolayer or alginate beads with and without PGE(2) and/or agonists of EP receptors, antagonists of EP receptors, and cytokines. Cell survival, proliferation, and total proteoglycan synthesis and accumulation were measured in alginate beads. Chondrocyte-related gene expression and phosphatidylinositol 3-kinase/Akt signaling were assessed by real-time reverse transcription-polymerase chain reaction and Western blotting, respectively, using a monolayer cell culture model. RESULTS: Stimulation of human articular chondrocytes with PGE(2) through the EP2 receptor suppressed proteoglycan accumulation and synthesis, suppressed aggrecan gene expression, did not appreciably affect expression of matrix-degrading enzymes, and decreased the type II collagen:type I collagen ratio. EP2 and EP4 receptors were expressed at higher levels in knee cartilage than in ankle cartilage and in a grade-dependent manner. PGE(2) titration combined with interleukin-1 (IL-1) synergistically accelerated expression of pain-associated molecules such as inducible nitric oxide synthase and IL-6. Finally, stimulation with exogenous PGE(2) or an EP2 receptor-specific agonist inhibited activation of Akt that was induced by insulin-like growth factor 1. CONCLUSION: PGE(2) exerts an antianabolic effect on human adult articular cartilage in vitro, and EP2 and EP4 receptor antagonists may represent effective therapeutic agents for the treatment of OA.

Critical role of vitamin D in sulfate homeostasis: regulation of the sodium-sulfate cotransporter by 1,25-dihydroxyvitamin D3.

As the fourth most abundant anion in the body, sulfate plays an essential role in numerous physiological processes. One key protein involved in transcellular transport of sulfate is the sodium-sulfate cotransporter NaSi-1, and previous studies suggest that vitamin D modulates sulfate homeostasis by regulating NaSi-1 expression. In the present study, we found that, in mice lacking the vitamin D receptor (VDR), NaSi-1 expression in the kidney was reduced by 72% but intestinal NaSi-1 levels remained unchanged. In connection with these findings, urinary sulfate excretion was increased by 42% whereas serum sulfate concentration was reduced by 50% in VDR knockout mice. Moreover, levels of hepatic glutathione and skeletal sulfated proteoglycans were also reduced by 18 and 45%, respectively, in the mutant mice. Similar results were observed in VDR knockout mice after their blood ionized calcium levels and rachitic bone phenotype were normalized by dietary means, indicating that vitamin D regulation of NaSi-1 expression and sulfate metabolism is independent of its role in calcium metabolism. Treatment of wild-type mice with 1,25-dihydroxyvitamin D3 or vitamin D analog markedly stimulated renal NaSi-1 mRNA expression. These data provide strong in vivo evidence that vitamin D plays a critical role in sulfate homeostasis. However, the observation that serum sulfate and skeletal proteoglycan levels in normocalcemic VDR knockout mice remained low in the absence of rickets and osteomalacia suggests that the contribution of sulfate deficiency to development of rickets and osteomalacia is minimal.

Spontaneous and experimental osteoarthritis in dog: similarities and differences in proteoglycan levels.

The unilateral canine model is the most commonly used model of experimental osteoarthritis (OA). In this model, the anterior cruciate ligament (ACL) of one knee is transected and the contralateral joint is usually used as a control. However, dogs, similar to humans, can develop OA spontaneously with old age. Additionally, certain breeds of dogs are genetically predisposed to OA and can develop symptoms at a young age. The goal of this study was to compare the pathological changes of proteoglycans in OA cartilage from dogs that developed OA spontaneously to those that underwent ACL transection. For this reason, biglycan, decorin and fibromodulin levels and degradation patterns were compared by Western blot hybridization, and aggrecan contents were quantified by dimethylmethylene blue assay. The changes in proteoglycan levels in the cartilage of dogs with spontaneous OA, regardless of their age, were very similar to those published for human OA cartilage. However, when OA developed as a result of ACL-surgery, the changes in proteoglycans were different from those of slowly developing spontaneous OA. Therefore, these differences should be taken into consideration when the ACL-transection model is used.

Genetic rescue of chondrodysplasia and the perinatal lethal effect of cartilage link protein deficiency.

The targeted disruption of cartilage link protein gene (Crtl1) in homozygous mice resulted in a severe chondrodysplasia and perinatal lethality. This raised the question of whether the abnormalities seen in Crtl1 null mice are all caused by the absence of link protein in cartilage or whether the deficiency of the protein in other tissues and organs contributed to the phenotype. To address this question we have generated transgenic mice overexpressing cartilage link protein under the control of a cartilage-specific promoter, and then these transgenic mice were used for a genetic rescue of abnormalities in Crtl1 null mice. While the overexpression of cartilage link protein resulted in no abnormal phenotype, the cartilage-specific transgene expression of link protein could completely prevent the perinatal mortality of link protein-deficient mice and, depending on the level of the link protein expression, rescue skeletal abnormalities. Although link protein was originally isolated from cartilage, we found and determined Crtl1 transcripts and corresponding proteins in every organ tested from mouse embryos to aging animals. We also identified three additional members of the link protein family, all co-localized with hyaluronic acid-binding proteoglycans in the mouse genome. The ubiquitous presence of link protein suggests a general and systemic function of link protein in the organization of extracellular matrix in a number of tissues, possibly interacting with other proteoglycans, such as versican, brevican, and neurocan.

Age-related changes in the proteoglycans of human skin. Specific cleavage of decorin to yield a major catabolic fragment in adult skin.

Dramatic changes occur in skin as a function of age, including changes in morphology, physiology, and mechanical properties. Changes in extracellular matrix molecules also occur, and these changes likely contribute to the overall age-related changes in the physical properties of skin. The major proteoglycans detected in extracts of human skin are decorin and versican. In addition, adult human skin contains a truncated form of decorin, whereas fetal skin contains virtually undetectable levels of this truncated decorin. Analysis of this molecule, herein referred to as decorunt, indicates that it is a catabolic fragment of decorin rather than a splice variant. With antibody probes to the core protein, decorunt is found to lack the carboxyl-terminal portion of decorin. Further analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry shows that the carboxyl terminus of decorunt is at Phe(170) of decorin. This result indicates that decorunt represents the amino-terminal 43% of the mature decorin molecule. Such a structure is inconsistent with alternative splicing of decorin and suggests that decorunt is a catabolic fragment of decorin. A neoepitope antiserum, anti-VRKVTF, was generated against the carboxyl terminus of decorunt. This antiserum does not recognize intact decorin in any skin proteoglycan sample tested on immunoblots but recognizes every sample of decorunt tested. The results with anti-VRKVTF confirm the identification of the carboxyl terminus of decorunt. Analysis of collagen binding by surface plasmon resonance indicates that the affinity of decorunt for type I collagen is 100-fold less than that of decorin. This observation correlates with the structural analysis of decorunt, in that it lacks regions of decorin previously shown to be important for interaction with type I collagen. The detection of a catabolic fragment of decorin suggests the existence of a specific catabolic pathway for this proteoglycan. Because of the capacity of decorin to influence collagen fibrillogenesis, catabolism of decorin may have important functional implications with respect to the dermal collagen network.

Changes in mRNA and protein levels of proteoglycans of the anulus fibrosus and nucleus pulposus during intervertebral disc degeneration.

STUDY DESIGN: This study correlates the mRNA and protein levels of large and small proteoglycans with the morphologic grade of degeneration. OBJECTIVES: To investigate changes in mRNA and protein levels of aggrecan, versican, biglycan, decorin and fibromodulin in the anulus fibrosus and the nucleus pulposus at different stages of tissue degeneration. SUMMARY OF BACKGROUND DATA: Proteoglycans are found in both the anulus fibrosus and nucleus pulposus and contribute to the hydration of the tissue (aggrecan) and the regulation of matrix assembly (small proteoglycans). Changes in their abundance may influence the biochemical and biomechanical properties of the disc and may have an effect on disc function. METHODS: Thirty-four human lumbar intervertebral discs were graded using Thompson's morphologic scale. The mRNA and protein content of aggrecan, versican, biglycan, decorin and fibromodulin was measured in extracts of anulus fibrosus and nucleus pulposus tissues dissected from these specimens. Protein levels were analyzed by Western blotting, and mRNA levels were semiquantified by reverse transcription polymerase chain reaction. RESULTS: In the anulus fibrosus, the mRNA and protein content of all proteoglycans was significantly elevated at early stages of degeneration then declined in severely degenerated tissues. In contrast, matrix components in the nucleus pulposus showed a continuous decrease in content with increasing grade of degeneration. CONCLUSIONS: Cells of the anulus fibrosus and nucleus pulposus react to tissue degeneration differently. Decreased mRNA expression by nucleus pulposus cells and declining protein content of the matrix make the nucleus more vulnerable to degeneration than the anulus. The cells in the anulus fibrosus respond to early degeneration by upregulating biosynthetic processes. However, in heavily degenerated tissues, the decline in the synthesis of aggrecan and the increase in the concentrations of small proteoglycans may be responsible for the failure of the repair processes.

The characterization of versican and its message in human articular cartilage and intervertebral disc.

Splicing variation of the versican message and size heterogeneity of the versican core protein were analyzed in human articular cartilage and intervertebral disc. Splicing variation of the message was studied by PCR analysis to detect the presence or absence of exons 7 and 8, which encode large chondroitin sulfate attachment regions. At all ages in normal cartilage from the third trimester fetus to the mature adult, the presence of the versican isoform possessing exon 8 but not exon 7 (V1) could be readily detected. The message isoforms possessing neither exon 7 nor 8 (V3) or both exons 7 and 8 (V0) were only detectable in the fetus, and the isoform possessing only exon 7 (V2) was never detected. In osteoarthritic cartilage and in adult intervertebral disc the versican message pattern was the same as that observed in the normal adult with only the isoform possessing exon 8 being detected. Core protein heterogeneity was studied by immunoblotting following enzymic removal of the glycosaminoglycan chains from the proteoglycan, using an antibody recognizing the globular G1 region of versican. All articular cartilage extracts from the fetus to the mature adult contained multiple core protein sizes of greater than 200 kDa. The adult cartilage extracts tended to have an increased proportion of the smaller sized core proteins and osteoarthritic cartilage possessed similar core protein sizes to the normal adult. In contrast, intervertebral disc at all post-natal ages showed a greater range of size heterogeneity with a prominent component of about 50 kDa. The abundance of this component increased if the samples were treated with keratanase prior to analysis, suggesting that the G1 region of versican in disc can be substituted with keratan sulfate. The increased presence of versican in the disc relative to articular cartilage may suggest a more pronounced functional role for this proteoglycan, particularly in the nucleus pulposus.