Cysteine-rich domain of type III collagen N-propeptide inhibits fibroblast activation by attenuating TGFß signaling.

TGFß is a key regulator of the dynamic reciprocity between cells and the extracellular matrix that drives physiologic and pathologic responses in both tissue repair and tumor microenvironments. Our studies define type III Collagen (Col3) as a suppressor of scar formation and desmoplasia through its effects, in part, on myofibroblasts. TGFß stimulates activation of myofibroblasts, and here, we demonstrate that cultured Col3-deficient fibroblasts have increased TGFß signaling compared to wild-type fibroblasts. Moreover, kinetic binding studies show that a synthetic peptide containing a Col3 cysteine-rich (CR) domain found within its N-propeptide binds in a dose-dependent manner to TGFß1, while a CR control peptide with mutated cysteines does not, suggesting that Col3 attenuates TGFß signaling in part through the N-propeptide CR domain. Consistent with this hypothesis, the CR peptide attenuates TGFß signaling in fibroblasts and 4T1 breast cancer cells and suppresses fibroblast activation and contraction, as assessed by α-smooth-muscle actin staining, cell wrinkling of deformable silicone, and stressed-fibroblast populated collagen lattice contraction assays. Finally, CR peptide treatment of orthotopically injected breast cancer cells (4T1) suppresses intratumoral fibroblast activation and inhibits primary tumor growth compared to CR control. Treatment with the CR peptide decreases both intratumoral canonical and non-canonical downstream TGFß signaling targets, consistent with its extracellular binding to TGFß. Taken together, our results suggest that the Col3 N-propeptide CR domain binds TGFß1 and attenuates (but importantly does not eliminate) TGFß signaling in fibroblasts and cancer cells. Expanding on our previous work, this study demonstrates an additional mechanism by which Col3 regulates cell behaviors in post-injury and tumor microenvironments and suggests that novel Col3-targeted strategies could effectively control biologic responses in vivo and improve anti-scarring/fibrosis and oncologic therapies.

Intervertebral Disc Cells Produce Interleukins Found in Patients with Back Pain.

OBJECTIVE: To examine the link between cytokines in intervertebral disc (IVD) tissues and axial back pain. DESIGN: In vitro study with human IVD cells cultured from cadaveric donors and annulus fibrosus (AF) tissues from patients. RESULTS: Cultured nucleus pulposus (NP) and AF cells were stimulated with interleukin (IL)-1ß. IL-8 and IL-7 gene expression was analyzed using real-time polymerase chain reaction. IL-8 protein was quantified by enzyme-linked immunosorbent assay. After IL-1ß stimulation, IL-8 gene expression increased 26,541 fold in NP cells and 22,429 fold in AF cells, whereas protein released by the NP and AF cells increased 2,389- and 1,784-fold, respectively. IL-7 gene expression increased 3.3-fold in NP cells (P < 0.05).Cytokine profiles in AF tissues collected from patients undergoing surgery for back pain (painful group) or scoliosis (controls) were compared by cytokine array. IL-8 protein in the AF tissues from patients with back pain was 1.81-fold of that in controls. IL-7 and IL-10 in AF tissues from the painful group were 6.87 and 4.63 times greater than the corresponding values in controls, respectively (P < 0.05). CONCLUSION: Inflammatory mediators found in AF tissues from patients with discogenic back pain are likely produced by IVD cells and may play a key role in back pain.

Fibronectin splice variation in human knee cartilage, meniscus and synovial membrane: observations in osteoarthritic knee.

Fibronectin (FN) is a widely expressed molecule that can participate in development of osteoarthritis (OA) affecting cartilage, meniscus, and synovial membrane (SM). The alternatively spliced isoforms of FN in joint tissues other than cartilage have not been extensively studied previously. The present study compares FN splice variation in patients with varying degrees of osteoarthritic change. Joint tissues were collected from asymptomatic donors and patients undergoing arthroscopic procedures. Total RNA was amplified by PCR using primers flanking alternatively spliced Extra Domain A (EDA), Extra Domain B (EDB) and Variable (V) regions. EDB(+) , EDB(-) and EDA(-) and V(+) variants were present in all joint tissues, while the EDA(+) variant was rarely detected. Expression levels of EDB(+) and EDV(+) variants were similar in cartilage, synovium, and meniscal tissues. Synovial expression of V(+) FN in arthroscopy patients varied with degree of cartilage degeneration. Two V(-) isoforms, previously identified in cartilage, were also present in SM and meniscus. Fibronectin splicing in meniscus and SM bears striking resemblance to that of cartilage. Expression levels of synovial V(+) FN varied with degree of cartilage degeneration. V(+) FN should be investigated as a potential biomarker of disease stage or progression in larger populations.

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.

Type III collagen regulates osteoblastogenesis and the quantity of trabecular bone.

Type III collagen (Col3), a fibril-forming collagen, is a major extracellular matrix component in a variety of internal organs and skin. It is also expressed at high levels during embryonic skeletal development and is expressed by osteoblasts in mature bone. Loss of function mutations in the gene encoding Col3 (Col3a1) are associated with vascular Ehlers-Danlos syndrome (EDS). Although the most significant clinical consequences of this syndrome are associated with catastrophic failure and impaired healing of soft tissues, several studies have documented skeletal abnormalities in vascular EDS patients. However, there are no reports of the role of Col3 deficiency on the murine skeleton. We compared craniofacial and skeletal phenotypes in young (6-8 weeks) and middle-aged (>1 year) control (Col3(+/+)) and haploinsufficient (Col3(+/-)) mice, as well as young null (Col3(-/-)) mice by microcomputed tomography (µCT). Although Col3(+/-) mice did not have significant craniofacial abnormalities based upon cranial morphometrics, µCT analysis of distal femur trabecular bone demonstrated significant reductions in bone volume (BV), bone volume fraction (BV/TV), connectivity density, structure model index and trabecular thickness in young adult female Col3(+/-) mice relative to wild-type littermates. The reduction in BV/TV persisted in female mice at 1 year of age. Next, we evaluated the role of Col3 in vitro. Osteogenesis assays revealed that cultures of mesenchymal progenitors collected from Col3(-/-) embryos display decreased alkaline phosphatase activity and reduced capacity to undergo mineralization. Consistent with this data, a reduction in expression of osteogenic markers (type I collagen, osteocalcin and bone sialoprotein) correlates with reduced bone Col3 expression in Col3(+/-) mice and with age in vivo. A small but significant reduction in osteoclast numbers was found in Col3(+/-) compared to Col3(+/+) bones. Taken together, these findings indicate that Col3 plays a role in development of trabecular bone through its effects on osteoblast differentiation.

Diminished type III collagen promotes myofibroblast differentiation and increases scar deposition in cutaneous wound healing.

The repair of cutaneous wounds in the postnatal animal is associated with the development of scar tissue. Directing cell activities to efficiently heal wounds while minimizing the development of scar tissue is a major goal of wound management and the focus of intensive research efforts. Type III collagen (Col3), expressed in early granulation tissue, has been proposed to play a prominent role in cutaneous wound repair, although little is known about its role in this process. To establish the role of Col3 in cutaneous wound repair, we examined the healing of excisional wounds in a previously described murine model of Col3 deficiency. Col3 deficiency (Col3+/-) in aged mice resulted in accelerated wound closure with increased wound contraction. In addition, Col3-deficient mice had increased myofibroblast density in the wound granulation tissue as evidenced by an increased expression of the myofibroblast marker, α-smooth muscle actin. In vitro, dermal fibroblasts obtained from Col3-deficient embryos (Col3+/- and -/-) were more efficient at collagen gel contraction and also displayed increased myofibroblast differentiation compared to those harvested from wild-type (Col3+/+) embryos. Finally, wounds from Col3-deficient mice also had significantly more scar tissue area on day 21 post-wounding compared to wild-type mice. The effect of Col3 expression on myofibroblast differentiation and scar formation in this model suggests a previously undefined role for this ECM protein in tissue regeneration and repair.

Fibronectin splicing variants in human intervertebral disc and association with disc degeneration.

STUDY DESIGN: In this study, normal intervertebral disc (IVD) tissues and degenerative human IVD tissues were compared for presence of fibronectin (FN) mRNA splice variants and for FN fragments (FN-f). OBJECTIVE: To further understand FN RNA splice forms and protein fragments in disc degeneration. SUMMARY OF BACKGROUND DATA: FN splice variants play important roles in regulating cell-matrix and matrix-matrix interactions in skeletogenesis and skeletal function in limbs and other sites. However, presence and possible roles of FN splice variants and fragments in human IVD have not been determined. METHODS: Normal infant and adult IVD tissues were obtained from organ donors, and degenerative human IVD tissues were obtained from patients undergoing spinal surgeries. FN splice patterns were assessed by reverse transcriptase polymerase chain reaction. Relative expression levels were semiquantified by densitometry. FN and its fragments were studied by Western blot analysis. RESULTS: Both the EDB and EDB splice variants were present in normal and degenerative IVD tissues. The EDB to EDB ratio was highest in moderately degenerative tissue. The EDA domain was only expressed in infant but not adult tissue. Variable-region (V) splice forms were present in all tissues studied. A splice form with the entire V-region, the 15th type III domain, and 10th type I domain adjacent to the 3'end of V region omitted (referred to as [V+III-15+I-10], also known as [V+C] splice form) was present at higher levels in adult than in infant samples. FN-f were also detected in degenerating tissue, but not in normal IVD tissue samples. CONCLUSION: The data indicate that higher levels of EDB isoform and FN-f are associated with IVD degeneration. This shift in alternative splicing may reflect an attempt of tissue repair and remodeling. Novel information gathered in this study will lead to a better understanding of pathologic processes associated with disc malfunction and degeneration.

Thyroid hormone treatment of cultured chondrocytes mimics in vivo stimulation of collagen X mRNA by increasing BMP 4 expression.

During endochondral bone formation, chondrocytes undergo terminal differentiation, during which the rate of proliferation decreases, cells become hypertrophic, and the extracellular matrix is altered by production of collagen X, as well as proteins required for matrix mineralization. This maturation process is responsible for most longitudinal bone growth, both during embryonic development and in postnatal long bone growth plates. Among the major signaling molecules implicated in regulation of this process are the positive regulators thyroid hormone (T3) and bone morphogenetic proteins (BMPs). Both T3 and BMPs are essential for endochondral bone formation and cannot compensate for each other, suggesting interaction of the two signaling pathways. We have analyzed the temporal and spatial expression patterns of numerous genes believed to play a role in chondrocyte maturation. Our results show that T3 stimulates collagen X gene expression in cultured chondrocytres with kinetics and magnitude similar to those observed in vivo. Stimulation of collagen X gene expression by T3 occurs only after a significant delay, implying that this hormone may act indirectly. We show further that T3 rapidly stimulates production of BMP 4, concomitant with a decrease in the BMP inhibitor Noggin, potentially resulting in a net increase in BMP signaling. Finally, inhibition of BMP signaling with exogenous Noggin prevents T3 stimulation of collagen X expression, indicating that BMP signaling is essential for this process. These data position thyroid hormone at the top of a T3/BMP cascade, potentially explaining why both pathways are essential for chondrocyte maturation. J. Cell. Physiol. 219: 595-605, 2009. (c) 2009 Wiley-Liss, Inc.

Integration of signaling pathways regulating chondrocyte differentiation during endochondral bone formation.

During endochondral bone formation, chondrocytes undergo a process of terminal differentiation or maturation, during which the rate of proliferation decreases, cells become hypertrophic, and the extracellular matrix is altered by production of a unique protein, collagen X, as well as proteins that promote mineralization. The matrix surrounding the hypertrophic chondrocytes eventually becomes mineralized, and the mineralized matrix serves as a template for bone deposition. This process is responsible for most longitudinal bone growth, both during embryonic development and in the postnatal long bone growth plates. Chondrocyte maturation must be precisely controlled, balancing proliferation with terminal differentiation; changes in the rate of either proliferation or differentiation result in shortened bones. Numerous signaling molecules have been implicated in regulation of this process. These include the negative regulators Indian hedgehog (Ihh) and parathyroid hormone-related protein (PTHrP; Pthlh, PTH-like hormone), as well as a number of positive regulators. This review will focus on several positive regulators which exert profound effects on chondrocyte maturation: the thyroid hormones T3 and T4, retinoic acid (the major active metabolite of vitamin A) and bone morphogenetic proteins (BMPs), as well as the transcription factor Runx2. Each of these molecules is essential for endochondral bone formation and cannot compensate for the others; abrogation of any one of them prevents differentiation. The important features of each of these signaling pathways will be discussed as they relate to chondrocyte maturation, and a model will be proposed suggesting how these pathways may converge to regulate this process.

Retinoids directly activate the collagen X promoter in prehypertrophic chondrocytes through a distal retinoic acid response element.

Retinoids are essential for the terminal differentiation of chondrocytes during endochondral bone formation. This maturation process is characterized by increased cell size, expression of a unique extracellular matrix protein, collagen X, and eventually by mineralization of the matrix. Retinoids stimulate chondrocyte maturation in cultured cells and experimental animals, as well as in clinical studies of synthetic retinoids; furthermore, retinoid antagonists prevent chondrocyte maturation in vivo. However, the mechanisms by which retinoids regulate this process are poorly understood. We and others showed previously that retinoic acid (RA) stimulates expression of genes encoding bone morphogenetic proteins (BMPs), suggesting that retinoid effects on chondrocyte maturation may be indirect. However, we now show that RA also directly stimulates transcription of the collagen X gene promoter. We have identified three RA response element (RARE) half-sites in the promoter, located 2,600 nucleotides upstream from the transcription start site. These three half-sites function as two overlapping RAREs that share the middle half-site. Ablation of the middle half-site destroys both elements, abolishing RA receptor (RAR) binding and drastically decreasing RA stimulation of transcription. Ablation of each of the other two half-sites destroys only one RARE, resulting in an intermediate level of RAR binding and transcriptional stimulation. These results, together with our previously published data, indicate that retinoids stimulate collagen X transcription both directly, through activation of RARs, and indirectly, through increased BMP production.

Nitric oxide-nitric oxide synthase regulates key maturational events during chondrocyte terminal differentiation.

The goal of this investigation was to explore the mechanism by which NOS and NO serve to regulate events linked to chondrocyte terminal differentiation. NOS isoform expression and NO adducts in chick growth cartilage were detected by immunohistochemistry and Western blot analysis. All NOS isoforms were expressed in chick growth plate chondrocytes with the highest levels present in the hypertrophic region. The enzymes were active since nitrosocysteine and nitrotyrosine residues were detected in regions of the epiphysis with the highest levels of NOS expression. Maturing chick sternal chondrocytes evidenced an increase in NO release and a rise in NOS protein levels. When treated with NOS inhibitors, there was a decrease in the alkaline phosphatase activity of the hypertrophic cells. On the other hand, NO donors caused a small but significant elevation in alkaline phosphatase activity. Transient transfections of chondrocytes with an endothelial NOS isoform caused an increase in collagen type X promoter activity. Induction of both collagen type X expression and alkaline phosphatase activity was blocked by inhibitors of the cGMP pathway. These findings indicate that NO is generated by three NOS isoforms in terminally differentiated chondrocytes. The expression of NOS and the generation of NO enhanced maturation by upregulating alkaline phosphatase and collagen type X expression. Since expression of these two determinants was blocked by inhibitors of the cGMP pathway, it is concluded that NO metabolism is required for development of the mature chondrocyte phenotype.

Stimulation of type-X collagen gene transcription by retinoids occurs in part through the BMP signaling pathway.

BACKGROUND: Chondrocyte maturation and hypertrophy during endochondral bone formation are stimulated by both retinoids and bone morphogenetic proteins (BMPs). The type-X collagen gene, which is expressed only in hypertrophic chondrocytes, provides an excellent marker for chondrocyte maturation. We previously identified a 651-base-pair region of the type-X collagen promoter that is essential for its activation by BMP. We examined the relationship between the retinoid and BMP signaling pathways in transcriptional stimulation of the type-X collagen gene to determine whether they act independently or interact to regulate endochondral bone formation. METHODS: Prehypertrophic chondrocytes from embryonic chick sterna cultured in the presence or absence of retinoic acid or BMP-2 were transiently transfected with plasmids containing various mutations of the type-X collagen promoter directing expression of a luciferase reporter gene. In addition, real-time polymerase chain reaction was used to examine the effects of retinoic acid on expression of genes encoding BMP-2, 4, and 6. RESULTS: The previously identified BMP-responsive region of the type-X collagen promoter also mediated stimulation by physiological concentrations of retinoic acid in prehypertrophic chondrocytes. Systematic deletion mutagenesis of the BMP/retinoid-responsive region of the type-X collagen promoter identified distinct regions that are responsible for promoter stimulation by retinoids and BMP. Retinoic acid rapidly and dramatically stimulated accumulation of BMP-2 and BMP-6 messenger RNAs. CONCLUSIONS: These results suggest that, while retinoic acid appears to stimulate type-X collagen gene transcription in part by stimulating the BMP signaling pathway, it also acts in part through mechanisms that are independent of BMP.

A novel noncollagenous protein encoded by an alternative transcript of the chick type III collagen gene is expressed in cartilage, bone and muscle.

We have identified a noncollagenous protein, Col3alt, encoded by an alternative transcript of the chick type III collagen gene; its amino acid sequence is out of frame with the collagen coding sequence. This 178-amino-acid protein is unique and has no recognizable motifs other than a hydrophobic domain. Col3alt is found in embryonic cartilage, muscle and bone and in the proliferative and prehypertrophic zones of juvenile chicken growth plates. The protein is intracellular in immature chondrocytes and myoblasts, but is extracellular in well-differentiated cartilage, muscle and bone, despite the lack of a conventional signal peptide. These results demonstrate an unexpected economy of genome utilization in which a single gene, using alternative promoters, gives rise to two unrelated proteins, type III collagen and Col3alt.