Sympathectomy aggravates subchondral bone changes during osteoarthritis progression in mice without affecting cartilage degeneration or synovial inflammation.

OBJECTIVE: Osteoarthritis (OA) pathogenesis involves the interaction of articular cartilage with surrounding tissues, which are innervated by tyrosine hydroxylase-positive (TH+) sympathetic nerve fibers suggesting a role of the sympathetic nervous system (SNS) during OA progression. We analyzed the effects of sympathectomy (Syx) in a murine OA model. METHODS: Peripheral Syx was generated by 6-hydroxydopamine (6-OHDA) injections in male C57BL/6 mice. OA was induced in wild-type (WT) and Syx mice by destabilization of the medial meniscus (DMM). TH+ fibers and splenic NE were analyzed to evaluate Syx efficiency. OA progression was examined by OARSI and synovitis scores and micro-CT. Expression of TH, α2A- and ß2-adrenergic receptors (AR), and activity of osteoblasts (ALP) and osteoclasts (TRAP) was investigated by stainings. RESULTS: Syx resulted in synovial TH+ fiber elimination and splenic NE decrease. Cartilage degradation and synovitis after DMM were comparably progressive in both WT and Syx mice. Calcified cartilage (CC) and subchondral bone plate (SCBP) thickness and bone volume fraction (BV/TV) increased in Syx mice due to increased ALP and decreased TRAP activities compared to WT 8 weeks after DMMWT and Syx mice developed osteophytes and meniscal ossicles without any differences between the groups. AR numbers decreased in cartilage but increased in synovium and osteophyte regions after DMM in both WT and Syx mice. CONCLUSION: Peripheral dampening of SNS activity aggravated OA-specific cartilage calcification and subchondral bone thickening but did not influence cartilage degradation and synovitis. Therefore, SNS might be an attractive target for the development of novel therapeutic strategies for pathologies of the subchondral bone.

[Research consortium Neuroimmunology and pain in the research network musculoskeletal diseases]. / Forschungsverbund Neuroimmunologie und Schmerz (Neuroimpa) im Forschungsnetz Muskuloskelettale Erkrankungen.

BACKGROUND: The research consortium Neuroimmunology and Pain (Neuroimpa) explores the importance of the relationships between the immune system and the nervous system in musculoskeletal diseases for the generation of pain and for the course of fracture healing and arthritis. MATERIAL AND METHODS: The spectrum of methods includes analyses at the single cell level, in vivo models of arthritis and fracture healing, imaging studies on brain function in animals and humans and analysis of data from patients. RESULTS: Proinflammatory cytokines significantly contribute to the generation of joint pain through neuronal cytokine receptors. Immune cells release opioid peptides which activate opioid receptors at peripheral nociceptors and thereby evoke hypoalgesia. The formation of new bone after fractures is significantly supported by the nervous system. The sympathetic nervous system promotes the development of immune-mediated arthritis. The studies show a significant analgesic potential of the neutralization of proinflammatory cytokines and of opioids which selectively inhibit peripheral neurons. Furthermore, they show that the modulation of neuronal mechanisms can beneficially influence the course of musculoskeletal diseases. DISCUSSION: Interventions in the interactions between the immune system and the nervous system hold a great therapeutic potential for the treatment of musculoskeletal diseases and pain.

Norepinephrine modulates osteoarthritic chondrocyte metabolism and inflammatory responses.

OBJECTIVE: Norepinephrine (NE) was measured in synovial fluid of trauma patients and sympathetic nerve fibers were detected in healthy and osteoarthritic (OA) joint tissues indicating that cartilage pathophysiology might be influenced by sympathetic neurotransmitters. The aim of this study was to elucidate the mostly unknown role of NE in OA chondrocyte metabolism and inflammatory responses. METHODS: Articular cartilage was received after total knee replacement surgery from OA patients. Expression of adrenergic receptors (AR) and tyrosine hydroxylase (TH) was tested with end point polymerase chain reaction (PCR) and immunohistochemistry. 3-dimensional (3D) cell cultures were employed to analyze effects of NE on chondrocyte cell metabolism and the expression of interleukins (ILs), matrix metalloproteases (MMPs), tissue inhibitor of metalloproteases (TIMPs), glycosaminoglycan (GAG) and collagen II under non- and inflammatory conditions. Chondrocyte monolayer cultures were used to specify AR subtypes, to analyze cell cycle distribution and to determine catecholamines in cell culture supernatants. RESULTS: AR subtypes and TH were detected in chondrocytes, whereas NE was not released in measurable amounts. 10(-6) M NE reversed IL-1ß induced changes in IL-8, MMP-13, GAG and collagen II expression/production indicating for ß-AR signaling. Additionally, NE caused cell cycle slow down and decreased proliferation via ß-AR signaling. 10(-8) M NE increased the number of proliferating cells and induced apoptosis via α1-AR signaling. CONCLUSIONS: NE affects chondrocytes from OA cartilage regarding inflammatory response and its cell metabolism in a dose dependent manner. The sympathetic nervous system (SNS) may have a dual function in OA pathology with preserving a stable chondrocyte phenotype via ß-AR signaling and OA pathogenesis accelerating effects via α-AR signaling.

The impact of chemical synovectomy with sodium morrhuate on human chondrocytes and cartilage in vitro.

The vessel sclerosing property of sodium morrhuate is useful in treatment of recurrent joint effusions particularly in cases of knee joint effusions. It also can be employed as an addition to surgical synovectomy. Little is known about the effects of this drug on cartilage. This study was designed to investigate the cytotoxic impact of sodium morrhuate on human chondrocytes and cartilage tissue in vitro. Primary chondrocytes from 13 patients were isolated and cultivated in three-dimensional alginate cultures. Furthermore, femoral cartilage explants of 10 patients were cultivated in vitro. Both chondrocytes and cartilage explants were exposed to mixture of sodium morrhuate and mepivacaine in different concentrations simulating chemical synovectomy. After 48 h, cell proliferation, viability, and cytotoxicity were measured. The cartilage specimens were analyzed for apoptosis by immunohistochemistry. Up to a dilution of 1:600, cells were found to be 100 % viable with a proliferation rate of 74 % compared to controls. From 1:400 onwards, a significant increase in LDH release was measured which reached at dilution of 1:200 74 % of high control, whereas histological examination showed no proof of apoptosis or necrosis in cartilage tissue. The results of this in vitro study demonstrate that the cytotoxic effects of sodium morrhuate on human chondrocytes, which lack their original extracellular matrix, manifest between dilutions of 1:500 and 1:400 and increase with higher concentrations of the drug. This effect was not found for cartilage explants, though.

[Cell-based therapy options for osteochondral defects. Autologous mesenchymal stem cells compared to autologous chondrocytes]. / Zellbasierte Therapieoptionen von Gelenkknorpeldefekten. Autologe mesenchymale Stammzellen vs. autologe Chondrozyten.

Cartilage defects are multifactorial and site-specific and therefore need a clear analysis of the underlying pathology as well as an individualized therapy so that cartilage repair lacks a one-for-all therapy. The results of comparative clinical studies using cultured chondrocytes in autologous chondrocyte implantation (ACI) have shown some superiority over conventional microfracturing under defined conditions, especially for medium or large defects and in long-term durability. Adult mesenchymal stem cells can be isolated from bone marrow, have the potency to proliferate in culture and are capable of differentiating into the chondrogenic pathway. They represent a promising versatile cell source for cartilage repair but the ideal conditions for cultivation and application in cartilage repair are not yet known or have not yet been characterized. Adding a scaffold offers mechanical stability and advances chondrogenic differentiation for both possible cell sources.

Biomechanical forces exert anabolic effects on osteoblasts by activation of SMAD 1/5/8 through type 1 BMP receptor.

Osteoblasts are mechanosensitive cells, which respond to biomechanical stimuli to regulate the bone structure through anabolic and catabolic gene regulation. To examine the effects of mechanical forces on the osteogenic responses through the SMAD signaling in osteoblasts, the cells were cultured in well-characterized mechanoresponsive 3-D scaffolds and exposed to 10% dynamic compressive strain (Cmp) at 1 Hz. Subsequently, SMAD phosphorylation and osteogenic gene induction was examined. Osteoblasts cultured in 3-D scaffolds exhibited increased constitutive SMAD 1/5/8 phosphorylation, as compared to monolayers cultures. This SMAD 1/5/8 phosphorylation was further upregulated after 10, 30 and 60 min in response to Cmp, exhibiting a peak activation at 30 min. No significant changes in SMAD2 phosphorylation were observed, suggesting signals generated by Cmp may not activate the Transforming Growth Factor-ß signaling cascade. Subsequently, biomechanical stimulation-induced SMAD 1/5/8 phosphorylation upregulated the expression of osteogenic genes such as Osteoprotegrin, Msx2 and Runx2. Dorsomorphin, a selective inhibitor of the bone morphogenetic protein (BMP) receptor type 1 (BMPR1), blocked Cmp-induced SMAD 1/5/8 phosphorylation, as well as Osteoprotegrin, Msx2 and Runx2 gene expression. Collectively, the present findings demonstrate that biomechanical stimulation of osteoblasts activates SMAD 1/5/8 in the BMP signaling pathway through BMPR1 and may enhance osteogenesis by upregulating SMAD-dependent osteogenic genes.

The transcription factor AP-2ɛ regulates CXCL1 during cartilage development and in osteoarthritis.

OBJECTIVE: Recently, the transcription factor AP-2ɛ was shown to be a regulator of hypertrophy in cartilage and to be differentially expressed in osteoarthritis (OA). However, the only known target gene of AP-2ɛ up to date is integrin alpha10. To better characterize the function of AP-2ɛ in cartilage we screened for additional target genes. DESIGN: Promoter analysis, ChIP-assays and electrophoretic mobility shift assay were used to characterize the regulation of a new AP-2ɛ target gene in detail. RESULTS: In this study, we determined the chemokine CXCL1, already known to be important in osteoarthritis (OA), as a new target gene of AP-2ɛ. We could confirm that CXCL1 is expressed in chondrocytes and significantly over-expressed in OA-chondrocytes. Transient transfection of chondrocytes with an AP-2ɛ expression construct led to a significant increase of the CXCL1 mRNA level in these cells. We identified three potential AP-2 binding sites within the CXCL1 promoter and performed luciferase assays, indicating that an AP-2 binding motif (AP-2.2) ranging from position -135 to -144 bp relative to the translation start is responsive to AP-2ɛ. This result was further addressed by site-directed mutagenesis demonstrating that activation of the CXCL1 promoter by AP-2ɛ is exclusively dependent on AP-2.2. Chromatin immunoprecipitation and electromobility shift assays confirmed the direct binding of AP-2ɛ to the CXCL1 promoter in OA-chondrocytes at this site. CONCLUSION: These findings revealed CXCL1 as a novel target gene of AP-2ɛ in chondrocytes and support the important role of AP-2ɛ in cartilage.

Enhanced cartilage regeneration in MIA/CD-RAP deficient mice.

Melanoma inhibitory activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP) is a small soluble protein secreted from chondrocytes. It was identified as the prototype of a family of extracellular proteins adopting an SH3 domain-like fold. In order to study the consequences of MIA/CD-RAP deficiency in detail we used mice with a targeted gene disruption of MIA/CD-RAP (MIA-/-) and analyzed cartilage organisation and differentiation in in vivo and in vitro models. Cartilage formation and regeneration was determined in models for osteoarthritis and fracture healing in vivo, in addition to in vitro studies using mesenchymal stem cells of MIA-/- mice. Interestingly, our data suggest enhanced chondrocytic regeneration in the MIA-/- mice, modulated by enhanced proliferation and delayed differentiation. Expression analysis of cartilage tissue derived from MIA-/- mice revealed strong downregulation of nuclear RNA-binding protein 54-kDa (p54(nrb)), a recently described modulator of Sox9 activity. In this study, we present p54(nrb) as a mediator of MIA/CD-RAP to promote chondrogenesis. Taken together, our data indicate that MIA/CD-RAP is required for differentiation in cartilage potentially by regulating signaling processes during differentiation.

Synergistic effects of growth and differentiation factor-5 (GDF-5) and insulin on expanded chondrocytes in a 3-D environment.

OBJECTIVE: To investigate the effects of growth and differentiation factor-5 (GDF-5) alone or in combination with insulin on engineered cartilage from primary or expanded chondrocytes during 3-dimensional in vitro culture. DESIGN: Juvenile bovine chondrocytes were seeded either as primary or as expanded (passage 2) cells onto polyglycolic acid fiber meshes and cultured for 3 weeks in vitro. Additionally, adult human chondrocytes were grown in pellet culture after expansion (passage 2). The culture medium was supplemented either with GDF-5 in varying concentrations or insulin alone, or with combinations thereof. RESULTS: For primary chondrocytes, the combination of GDF-5 and insulin led to increased proliferation and construct weight, as compared to either factor alone, however, the production of glycosaminoglycans (GAG) and collagen per cell were not affected. With expanded bovine chondrocytes, the use of GDF-5 or insulin alone led to only very small constructs with no type II collagen detectable. However, the combination of GDF-5 (0.01 or 0.1 microg/ml) and insulin (2.5 microg/ml) yielded cartilaginous constructs and, in contrast to the primary cells, the observed redifferentiating effects were elicited on the cellular level independent of proliferation (increased production of GAG and collagen per cell, clear shift in collagen subtype expression with type II collagen observed throughout the construct). The synergistic redifferentiating effects of the GDF-5/insulin combination were confirmed with expanded adult human cells, also exhibiting a clear shift in collagen subtype expression on the mRNA and protein level. CONCLUSIONS: In combination with insulin, GDF-5 appears to enable the redifferentiation of expanded chondrocytes and the concurrent generation of cartilaginous constructs. The demonstration of these synergistic effects also for adult human chondrocytes supports the clinical relevance of the findings.

A muCT analysis of the femoral bone stock in osteonecrosis of the femoral head compared to osteoarthrosis.

INTRODUCTION: Long-term results of resurfacing in osteonecrosis of the femoral head (ONFH) are lacking and migration of the femoral component, necrosis of the femoral head and fracture of the femoral neck or head have been depicted. The aim of this study was to analyse the bone quality and 3-dimensional microarchitecture of the femoral head and neck in patients with ONFH compared to a group of patients with primary osteoarthritis (OA). MATERIALS AND METHODS: muCT was used to compare the microarchitecture of the femoral head and neck in patients with ONFH (n = 10) and a control group of patients with primary OA (n = 10). RESULTS: No statistical significant differences were found for bone volume and the bone volume fraction, the connectivity density and the structure model index in patients with ONFH and those with OA (P > 0.05). Patients with ONFH and those with OA had a similar trabecular number, thickness and separation (P > 0.05). CONCLUSION: Based on our findings, we might conclude that hip resurfacing arthroplasty is a possible therapeutic option in the treatment of ONFH of the young and active patient and that an insufficient bone stock in ONFH seems not to be the deciding factor for failure. However, we just focussed on one of probably various factors. Further studies are needed to support these findings.

Differential transcriptome analysis of intraarticular lesional vs intact cartilage reveals new candidate genes in osteoarthritis pathophysiology.

OBJECTIVE: To elucidate disease-specific molecular changes in osteoarthritis (OA) by analyzing the differential gene expression profile of damaged vs intact cartilage areas within the same joint of patients with OA of the knee using a combination of a novel RNA extraction technique and whole-genome oligonucleotide arrays. METHODS: The transcriptome of macroscopically affected vs intact articular cartilage as determined by visual assessment was analyzed using an optimized mill-based total RNA isolation directly from the tissue and high density synthetic oligonucleotide arrays. Articular cartilage samples were obtained from patients with OA of the knee. Expression of differentially regulated genes was validated by real-time quantitative polymerase chain reaction and immunohistochemistry. RESULTS: The amount of RNA obtained by the optimized extraction procedure was at least 1 microg per 500 mg of cartilage and fulfilled the common quality requirements. After hybridization onto HG-U133 Plus 2.0 GeneChips (Affymetrix), 28.6-51.7% of the probe sets on the microarray showed a detectable signal above the signal threshold in the individual samples. A subset of 411 transcripts, which appeared to be differentially expressed, was obtained when applying predefined filtering criteria. Of these, six genes were found to be up-regulated in the affected cartilage of all patients, including insulin-like growth factor binding protein 3 (IGFBP-3), wnt-1-inducible signaling protein 1 (WISP-1), aquaporin 1 (AQP-1), delta/notch-like EGF-repeat containing transmembrane (DNER), decay accelerating factor (DAF), complement factor I (IF). CONCLUSION: The optimized methodical approach reported here not only allows to determine area-specific gene expression profiles of intraindividually different low-RNA containing OA cartilage specimens. In addition, this study also revealed novel genes not yet reported to play a role in the pathophysiology of joint destruction in OA.

[Use of bone marrow mesenchymal stem cells for ex vivo cartilage regeneration]. / Einsatz von mesenchymalen Knochenmarkstammzellen für die Ex-vivo-Knorpelregeneration.

Articular cartilage disorders and injuries often result in lifelong chronic pain and compromised quality of life. When it comes to local articular cartilage defects, modern medicine is limited to short-term pain relief and inflammation control. In extreme cases the affected tissue is surgically removed and replaced by a synthetic prosthesis of limited durability. Cell-based therapies to regenerate articular cartilage have been in use since 1994. Such therapies provide a healthy population of cells to the injured site and require differentiated chondrocytes from the uninjured site as base material. Their usage often leads to donor site morbidity and they generate rigid fibrous cartilage where more flexible hyaline cartilage is required. The major restrictive factors for such methods are inadequate number and limited proliferation capacity of chondrocytes in vitro. Tissue engineering of adult marrow stromal cells/mesenchymal stem cells (MSCs) with their almost unlimited proliferation potential and proven capability to differentiate into chondrocytes for ex vivo generation of cartilage tissue still remains a vision. For optimal harnessing of MSCs as chondroprogenitor cells, basic background information regarding commitment to the lineage, cartilage differentiation and the regulatory factors and molecules involved is essential.

Effect of hypoxia and reoxygenation on gene expression and response to interleukin-1 in cultured articular chondrocytes.

OBJECTIVE: To determine the effects of hypoxia and reoxygenation on the metabolism of chondrocytes and their response to interleukin-1beta (IL-1beta). The study included activation of hypoxia-inducible factor 1 (HIF-1), NF-kappaB, and activator protein 1 (AP-1) transcription factors, expression of matrix components and metalloproteases and transforming growth factor beta (TGFbeta) and TGFbeta receptors, and production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)). METHODS: Bovine articular chondrocytes (BACs) were cultured to confluency in either 5% O(2) (hypoxia) or 21% O(2) (normoxia) in media supplemented with 10% fetal calf serum (FCS). BACs were preincubated for 18 hours in media with 1% FCS only and then incubated for 24 hours in the presence of IL-1beta. For reoxygenation experiments, cells were treated in the same way in 5% O(2), except that cultures were transferred to normal atmospheric conditions and used after 4 hours for RNA extraction or after 30 minutes for cytoplasmic or nuclear protein extraction. RESULTS: In hypoxic and reoxygenated chondrocytes, we observed strong DNA binding of HIF-1. IL-1beta-induced DNA binding of NF-kappaB and AP-1 was significantly higher in hypoxic and reoxygenated cultures than in normoxia. Greater activation of the MAPKs was also observed with IL-1beta treatment in hypoxia compared with normoxia. Steady-state levels of type II collagen and aggrecan core protein messenger RNA (mRNA) were decreased by IL-1beta in all instances. Matrix metalloprotease 1 (MMP-1) and MMP-3 mRNA were increased by IL-1beta in normoxia and hypoxia, whereas only MMP-3 mRNA was enhanced in reoxygenated cultures. The MMP-2 mRNA level was not significantly affected by IL-1beta in normoxia or hypoxia, whereas it was enhanced in reoxygenated cultures. MMP-9 mRNA was dramatically decreased by IL-1beta only in low oxygen tension. Tissue inhibitor of metalloproteinases 1 (TIMP-1) message was significantly enhanced by the cytokine in most instances, whereas TIMP-2 message was markedly decreased by IL-1beta in reoxygenated cultures. Stimulation of TGFbeta1 expression by IL-1beta was observed only in normal atmospheric conditions. One of the more striking findings of the study was the greater stimulating effect of IL-1beta on NO production observed in hypoxia, which was much higher than in normoxia, whereas the reverse was observed for IL-1beta-induced PGE(2) production. CONCLUSION: Oxygen level and reoxygenation stress significantly modulate gene expression and the response of articular chondrocytes to cytokines such as IL-1beta. In hypoxic conditions, which mimic the in vivo condition of cartilage, the effects of IL-1beta on both synthesis and degradative processes are significantly different from those in normoxia, conditions that are unlikely encountered by chondrocytes in a normal state. In low oxygen tension, high IL-1beta-induced NO production is associated with a significant decrease in PGE(2) synthesis. These data should influence our concept of the role of oxygen in the pathophysiology of joint disease and may help define the best conditions in which to develop bioartificial cartilage.

Paracrine interactions of chondrocytes and macrophages in cartilage degradation: articular chondrocytes provide factors that activate macrophage-derived pro-gelatinase B (pro-MMP-9).

Cells of the monocyte/macrophage lineage are involved in the development of inflammatory joint diseases such as rheumatoid arthritis. This disease is characterized by cartilage degradation and synovial membrane inflammation with a progressive loss of joint function. The pathological processes are still not well understood. Therefore it would be interesting to develop a suitable experimental in vitro model system for defined studies of monocyte/macrophage and chondrocyte interactions at the molecular level. For that purpose we cocultured chondrocytes from adult human articular cartilage with human monocytes and macrophages for defined periods of time in agarose without addition of serum. We performed zymographic and western blot analysis of culture medium, completed by quantitative RT-PCR of each chondrocyte, monocyte and macrophage RNA, respectively. The reliability of the newly established coculture systems is confirmed by causing a clear decrease of intact aggrecan in the coculture medium plus concurrent appearance of additional smaller fragments and a reduction of chondrocyte aggrecan and collagen II gene expression in the presence of monocytes. In culture medium from cocultures we detected active forms of the matrix metalloproteinases MMP-1, MMP-3 and MMP-9 accompanied by induction of gene expression of MMP-1, membrane type 1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in chondrocytes. No gene expression of MMP-9 was detectable in chondrocytes, the enzyme was solely expressed in monocytes and macrophages and was downregulated in the presence of chondrocytes. Our results suggest that MMP-9 protein in coculture medium originated from monocytes and macrophages but activation required chondrocyte-derived factors. Because addition of plasmin, a partial activator of pro-MMP-3 and pro-MMP-1, enhanced the activation of pro-MMP-9 and pro-MMP-1 in cocultures but not in monocultured macrophages, and the presence of MMP-3 inhibitor II prevented pro-MMP-9 activation, we assumed a stepwise activation process of pro-MMP-9 that is dependent on the presence of at least MMP-3 and possibly also MMP-1.

Alteration of fracture stability influences chondrogenesis, osteogenesis and immigration of macrophages.

Mechanical conditions at the fracture line determine the mode of fracture healing (osteonal versus non-osteonal bone union). The aim of this study was to investigate the influence of differing degrees of fracture stability on the time course of chondrogenesis, enchondral ossification and immigration of macrophages into the fracture callus. Using a fracture model of the rat's tibia, histological (Azan staining), immunohistological (antibodies directed against the macrophage-specific surface antigen ED2), and molecular biological techniques (expression of the mRNA of the cartilage-specific collagen IX, osteocalcin - a marker for mature osteoblasts - and the macrophage-specific macrosialin) were employed. In terms of histology and molecular biology (collagen IX mRNA expression) chondrogenesis in the fracture gap continued for longer in less stable fractures. In more stable fractures bone formation - identified by osteocalcin mRNA expression - increased from day 12 onwards. The expression of the macrophage-specific surface antigen ED2 and the mRNA of macrosialin was more pronounced but of shorter duration in the more stable fractures. This study shows that differing degrees of fracture stability not only influence the interplay between osteogenesis and chondrogenesis but also alter the kinetics of macrophage immigration into the fracture callus. These findings could aid in better understanding the cytobiologic mechanisms of callus formation and may suggest that macrophages are an important factor not only in soft tissue healing but also in bone healing.

Collagen XVI is expressed by human dermal fibroblasts and keratinocytes and is associated with the microfibrillar apparatus in the upper papillary dermis.

Indirect immunofluorescence staining of normal skin with affinity-purified antibodies revealed a conspicuous presence of collagen XVI at the dermo-epidermal interface where it occurs in close vicinity to collagen VII. In addition, the protein co-localizes with fibrillin 1 at the cutaneous basement membrane zone and the adjacent papillary dermis, but not in deeper layers of the dermis. Both fibronectin and collagen XVI are distributed throughout smooth muscles of hair follicles but do not co-localize. These data suggest, therefore, that collagen XVI contributes to the structural integrity of the dermo-epidermal junction zone by interacting with components of the anchoring complexes and the microfibrillar apparatus. A strong immunofluorescence signal associated with the extracellular matrix of individual cells was observed for keratinocytes or fibroblasts in monolayer cultures. Therefore, both cell types are likely sources of the protein also in situ. The rate of expression of collagen XVI mRNA in keratinocytes is about half of that in normal human skin fibroblasts. In both cell types, TGF-beta2 treatment results in an up-regulation of the collagen XVI-mRNA by approximately 50%. In keratinocytes, synthesis of collagen XVI protein and deposition to the cell layer and the extracellular matrix is stimulated fivefold and twofold, respectively. Since TGF-beta2 also upregulates the biosynthesis of other matrix macromolecules in the subepidermal zone the factor is likely to contribute to the stabilization of matrix zones near basement membranes in healing wounds.

Mouse fibulin-2 gene. Complete exon-intron organization and promoter characterization.

Fibulin-2, an extracellular matrix protein containing tandem arrays of calcium-binding epidermal growth factor-like motifs, is present in the basement membrane and stroma of many tissues. Its expression pattern suggested an essential role in organogenesis, particularly in embryonic heart development. In this study, we cloned the extreme 5' end of the mouse fibulin-2 cDNA, isolated phage and cosmid clones encoding the entire gene, and functionally characterized the promoter. The gene was found to consist of 18 exons spanning 55 kb of DNA. The exon-intron organization reflected the modular structure of the protein. Exon 9 was subjected to alternative splicing. All splice junctions conformed to the GT/AG rule, except that GC instead of GT was found in the splice donor site of exon 4. The gene lacked TATA and CAAT boxes but contained an initiator element (Inr) and several consensus Sp1 binding sites surrounding the transcription start sites. By transient transfection of promoter deletion constructs, a 0.46-kb region containing the clustered Sp1 sites was found to confer a high promoter activity.

Collagen type XVI expression is modulated by basic fibroblast growth factor and transforming growth factor-beta.

We investigated the effects of bFGF and TGF-beta on the expression of type XVI collagen, a member of the fibril associated collagen family, in human dermal fibroblasts and arterial smooth muscle cells. We found that bFGF decreased the alpha1(XVI) collagen mRNA to 18-24% of the controls, while TGF-beta increased the mRNA to 150-360%. Immunoprecipitation of metabolically labeled cells revealed corresponding, but less pronounced, changes at the protein levels. The results suggested that type XVI collagen expression is regulated by bFGF and TGF-beta in a manner similar to their regulation of the major type I fibrillar collagen produced by these cells.

Biosynthesis and processing of type XVI collagen in human fibroblasts and smooth muscle cells.

The alpha 1(XVI) collagen chain, recently identified by cDNA cloning, exhibits structural similarity to a subgroup of collagens that associate with collagen fibrils. Recombinant alpha 1(XVI) collagen chains produced in embryonic kidney cells are able to form stable homotrimers, which are rapidly converted into smaller polypeptides after secretion into the culture medium. In this study, we investigated the biosynthesis of native type XVI collagen by immunoprecipitation of metabolically labeled human cells. Dermal fibroblasts and arterial smooth muscle cells were precipitated with three antibodies raised against distinct regions in the N- and C-terminal part of the human alpha 1(XVI) collagen chain. A disulfide-bonded polypeptide of 220 kDa was obtained from the culture medium, cells and extracellular matrix with all three antibodies. This polypeptide is sensitive to bacterial collagenase digestion and partially resistant to pepsin digestion, suggesting that it is the endogenous alpha 1(XVI) collagen chain. Pulse/chase experiments showed that the newly synthesized alpha 1(XVI) chains are secreted into the medium and deposited in the extracellular matrix in a time-dependent manner. Unlike the recombinant chain, the native type XVI collagen does not undergo extensive proteolytic processing upon secretion. Both cell types deposit a substantial amount of the newly synthesized alpha 1(XVI) chain into the extracellular matrix, in which the 220-kDa polypeptide is the only product immunoprecipitated. There is little evidence for the presence of another constituent chain. The data are consistent with a nomotrimeric chain composition for type XVI collagen. No apparent difference exists in the rate of synthesis and secretion between fibroblasts and smooth muscle cells. Indirect immunofluorescence microscopy showed an extracellular distribution of type XVI collagen, which is located close to cells but not associated with fibrillar structures.