Dominik R. Haudenschild

Interleukin-17 family and IL-17 receptors

Interleukin-17 (IL-17) is a pro-inflammatory cytokine secreted by activated T-cells. Recently discovered related molecules are forming a family of cytokines, the IL-17 family. The prototype member of the family has been designated IL-17A. Due to recent advances in the human genome sequencing and proteomics five additional members have been identified and cloned: IL-17B, IL-17C, IL-17D, IL-17E and IL-17F. The cognate receptors for the IL-17 family identified thus far are: IL-17R, IL-17RH1, IL-17RL (receptor like), IL-17RD and IL-17RE. However, the ligand specificities of many of these receptors have not been established. The IL-17 signaling system is operative in disparate tissues such as articular cartilage, bone, meniscus, brain, hematopoietic tissue, kidney, lung, skin and intestine. Thus, the evolving IL-17 family of ligands and receptors may play an important role in the homeostasis of tissues in health and disease beyond the immune system. This survey reviews the biological actions of IL-17 signaling in cancers, musculoskeletal tissues, the immune system and other tissues.

Musculoskeletal changes following non-invasive knee injury using a novel mouse model of post-traumatic osteoarthritis

OBJECTIVE Post-traumatic osteoarthritis (PTOA) is a common consequence of traumatic joint injury, with 50% of anterior cruciate ligament (ACL) rupture patients developing PTOA within 10-20 years. Currently accepted mouse models of PTOA initiate symptoms using various methods, none of which faithfully mimic clinically-relevant injury conditions. In this study we characterize a novel non-invasive mouse model of PTOA that injures the ACL with a single load of tibial compression overload. We utilize this model to determine the time course of articular cartilage and subchondral bone changes following knee injury. DESIGN Mice were euthanized 1, 3, 7, 14, 28, or 56 days after non-invasive knee injury. Knees were scanned using micro-computed tomography (μCT) in order to quantify subchondral trabecular bone, subchondral bone plate, and non-native bone formation (heterotopic ossification). Development of osteoarthritis (OA) was graded using the osteoarthritis research society international (OARSI) scale on histological sections of injured and uninjured knees. RESULTS Following injury we observed a rapid loss of trabecular bone in injured knees compared to uninjured knees by 7 days post-injury, followed by a partial recovery of trabecular bone to a new steady state by 28 days post-injury. We also observed considerable non-native bone formation by 56 days post-injury. Grading of histological sections revealed deterioration of articular cartilage by 56 days post-injury, consistent with development of mild OA. CONCLUSIONS This study establishes a novel mouse model of PTOA, and describes the time course of musculoskeletal changes following knee injury, helping to establish the window of opportunity for preventative treatment.

Bone Morphogenetic Protein (BMP)-6 Signaling and BMP Antagonist Noggin in Prostate Cancer

It has been proposed that the osteoblastic nature of prostate cancer skeletal metastases is due in part to elevated activity of bone morphogenetic proteins (BMPs). BMPs are osteoinductive morphogens, and elevated expression of BMP-6 correlates with skeletal metastases of prostate cancer. In this study, we investigated the expression levels of BMPs and their modulators in prostate, using microarray analysis of cell cultures and gene expression. Addition of exogenous BMP-6 to DU-145 prostate cancer cell cultures inhibited their growth by up-regulation of several cyclin-dependent kinase inhibitors such as p21/CIP, p18, and p19. Expression of noggin, a BMP antagonist, was significantly up-regulated by BMP-6 by microarray analysis and was confirmed by quantitative reverse transcription-polymerase chain reaction and at the protein level. Noggin protein was present in prostate biopsies and localized to the epithelial components of prostate by immunohistochemistry. Recombinant noggin inhibited the function of BMP-6, suggesting a negative feedback regulation of BMP activity and indicating a strategy for the development of a novel therapeutic target in the treatment of painful osteosclerotic bone metastases of prostate cancer.

Rho kinase-dependent activation of SOX9 in chondrocytes.

OBJECTIVE The transcription factor SOX9 directly regulates the expression of the major proteoglycans and collagens comprising the cartilage extracellular matrix. The DNA binding activity and cellular localization of SOX9 is controlled through posttranslational modifications, including phosphorylation. The activity of Rho kinase (ROCK) has profound effects on the actin cytoskeleton, and these effects are instrumental in determining the phenotype and differentiation of chondrocytes. However, the mechanisms linking ROCK to altered chondrocyte gene expression remain unknown. The purpose of the present study was to test for a direct interaction between ROCK and SOX9. METHODS Human SW1353 chondrosarcoma cells were transfected with constructs coding for RhoA, ROCK, Lim kinase, and SOX9. The interaction between ROCK and SOX9 was tested on purified proteins, and was verified within a cellular context using induced overexpression and activation of the Rho pathway. The effects of SOX9 transcriptional activation were quantified with a luciferase reporter plasmid containing SOX9 binding sites from the COL2A1 enhancer element. RESULTS SOX9 was found to contain a consensus phosphorylation site for ROCK. In vitro, ROCK directly phosphorylated SOX9 at Ser(181), and the overexpression of ROCK or the activation of the RhoA pathway in SW1353 chondrosarcoma cells increased SOX9(Ser181) phosphorylation. ROCK caused a dose-dependent increase in the transcription of a SOX9-luciferase reporter construct, and increased phosphorylation and nuclear accumulation of SOX9 protein in response to transforming growth factor beta treatment and mechanical compression. CONCLUSION These results demonstrate a new interaction that directly links ROCK to increased cartilage matrix production via activation of SOX9 in response to mechanical and growth factor stimulation.

Vimentin contributes to changes in chondrocyte stiffness in osteoarthritis.

Actin and tubulin cytoskeletal components are studied extensively in chondrocytes, but less is known about vimentin intermediate filaments. In other cell types, vimentin is a determinant of cell stiffness and disruption of vimentin networks weakens the mechanical integrity of cells. Changes in vimentin organization correlate with osteoarthritis progression, but the functional consequences of these changes remain undetermined in chondrocytes. The objective of this study was to compare the contribution of vimentin to the mechanical stiffness of primary human chondrocytes isolated from normal versus osteoarthritic cartilage. Chondrocytes were embedded in alginate and vimentin networks disrupted with acrylamide. Constructs were imaged while subjected to 20% nominal strain on a confocal microscope stage, and the aspect ratios of approximately 1,900 cells were measured. Cytosolic stiffness was estimated with a finite element model, and live‐cell imaging of GFP‐vimentin was used to further analyze the nature of vimentin disruption. Vimentin in normal chondrocytes formed an inner cage‐like network that was substantially stiffer than the rest of the cytosol and contributed significantly to overall cellular stiffness. Disruption of vimentin reduced stiffness approximately 2.8‐fold in normal chondrocytes. In contrast, osteoarthritic chondrocytes were less stiff and less affected by vimentin disruption. This 3D experimental system revealed contributions of vimentin to chondrocyte stiffness previously not apparent, and correlated changes in vimentin‐based chondrocyte stiffness with osteoarthritis.

Interleukin-17 receptor-like gene is a novel antiapoptotic gene highly expressed in androgen-independent prostate cancer.

We have recently identified a new gene, interleukin-17 receptor-like (IL-17RL), which is expressed in normal prostate and prostate cancer. This investigation is focused on the role of IL-17RL in prostate cancer. We found that IL-17RL was expressed at significantly higher levels in several androgen-independent prostate cancer cell lines (PC3, DU145, cds1, cds2, and cds3) and tumors compared with the androgen-dependent cell lines (LNCaP and MLC-SV40) and tumors. In an in vivo model of human prostate tumor growth in nude mice (CWR22 xenograft model), IL-17RL expression in tumors was induced by androgen deprivation. The relapsed androgen-independent tumors expressed higher levels of IL-17RL compared with the androgen-dependent tumors. Overexpression of IL-17RL in tumor necrosis factor alpha (TNFalpha)-sensitive LNCaP cells inhibited TNFalpha-induced apoptosis by blocking activation of caspase-3 downstream to caspase-2 and caspase-8. Reciprocally, knocking down IL-17RL expression by small interfering RNA induced apoptosis in all the prostate cancer cell lines studied. Taken together, these results show that IL-17RL is a novel antiapoptotic gene, which may confer partially the property of androgen-independent growth of prostate cancer by promoting cell survival. Thus, IL-17RL is a potential therapeutic target in the treatment of prostate cancer.

Differential expression of multiple genes during articular chondrocyte redifferentiation.

Articular chondrocytes undergo a rapid change in phenotype and gene expression, termed dedifferentiation, when isolated from cartilage tissue and cultured on tissue culture plastic. On the other hand, “redifferentiation” of articular chondrocytes in suspension culture is characterized by decreased cellular proliferation and the reinitiation of synthesis of hyaline articular cartilage extracellular matrix molecules. The molecular triggers for these events have yet to be defined. Subtracted cDNA libraries representing genes involved in the early events of adult human articular chondrocyte redifferentiation were generated from human articular chondrocytes that were first cultured in monolayer, and subsequently transferred to suspension culture at 106 cells/ml for redifferentiation. Differential regulation of genes involved in cellular organization, nuclear structure, cellular growth regulation, and extracellular matrix deposition and remodeling were observed within 48 hr of this transfer. Many of these genes had not been previously identified in the chondrocyte differentiation pathway and a number of the isolated cDNAs did not have homologies to sequences in the public data banks. Genes involved in IL‐6 signal transduction including acute phase response factor (APRF), Mn superoxide dismutase, and IL‐6 itself were up‐regulated in suspension culture. Membrane glycoprotein gp130, a component of the IL‐6 receptor, was down‐regulated. Other genes involved in cell polarity, cell adherence, apoptosis, and possibly TGF‐beta signaling were differentially regulated. The differential regulation of the cytokine connective tissue growth factor (CTGF) during the early stages of articular chondrocyte redifferentiation, decreasing within 48 hours of transfer to suspension culture, was particularly interesting given its reported role in the stimulation of cellular proliferation. CTGF was highly expressed in proliferative monolayer culture, and then greatly reduced by redifferentiation in standard high‐density suspension culture. When articular chondrocytes were seeded in suspension at low‐density (104 cells/ml), however, high levels of CTGF were observed along with increased levels of mature articular cartilage extracellular matrix protein RNAs, such as type II collagen and aggrecan. Although the role of CTGF in articular cartilage biology remains to be elucidated, the results described here demonstrate the potential utility of subtractive hybridization in understanding the process of articular chondrocyte redifferentiation. Anat Rec 263:91–98, 2001.

Immunolocalization of IL-17A, IL-17B, and Their Receptors in Chondrocytes During Fracture Healing

Fracture healing in long bones is a sequential multistep cascade of hemostasis, transient inflammation, chemotaxis of progenitor cells, mitosis, differentiation of cartilage, and replacement with bone. This multistep cascade is orchestrated by cytokines and morphogens. Members of the interleukin (IL)-17 family, including IL-17B, have been identified in cartilage, but their expression during fracture healing is unknown. In this study, we determined the immunolocalization of cytokines IL-17A and IL-17B, along with the IL-17 receptor (IL-17R) and IL-17 receptor-like protein (IL-17RL), during the sequence of fracture repair in a standard model. The results were extended to developmental changes in the epiphyseal growth plate of long bones. Members of the IL-17 family were localized in chondrocytes in the fracture callus. Moreover, we found significant parallels to the localization of these cytokines and their receptors in chondrocytes during an endochondral differentiation program in the epiphyseal growth plate.

Non-invasive mouse models of post-traumatic osteoarthritis

Animal models of osteoarthritis (OA) are essential tools for investigating the development of the disease on a more rapid timeline than human OA. Mice are particularly useful due to the plethora of genetically modified or inbred mouse strains available. The majority of available mouse models of OA use a joint injury or other acute insult to initiate joint degeneration, representing post-traumatic osteoarthritis (PTOA). However, no consensus exists on which injury methods are most translatable to human OA. Currently, surgical injury methods are most commonly used for studies of OA in mice; however, these methods may have confounding effects due to the surgical/invasive injury procedure itself, rather than the targeted joint injury. Non-invasive injury methods avoid this complication by mechanically inducing a joint injury externally, without breaking the skin or disrupting the joint. In this regard, non-invasive injury models may be crucial for investigating early adaptive processes initiated at the time of injury, and may be more representative of human OA in which injury is induced mechanically. A small number of non-invasive mouse models of PTOA have been described within the last few years, including intra-articular fracture of tibial subchondral bone, cyclic tibial compression loading of articular cartilage, and anterior cruciate ligament (ACL) rupture via tibial compression overload. This review describes the methods used to induce joint injury in each of these non-invasive models, and presents the findings of studies utilizing these models. Altogether, these non-invasive mouse models represent a unique and important spectrum of animal models for studying different aspects of PTOA.

Enhanced activity of transforming growth factor β1 (TGF-β1) bound to cartilage oligomeric matrix protein.

Background: Cartilage oligomeric matrix protein (COMP) is a cartilage protein with a repeated modular structure that binds to and assembles extracellular matrix proteins but is not known to bind growth factors. Results: TGF-β family growth factors bind to multiple sites on COMP. Binding enhances TGF-β-dependent gene expression. Conclusion: COMP enhances the cellular response to TGF-β. Significance: A new function of COMP is to modulate TGF-β activity. Cartilage oligomeric matrix protein (COMP) is an important non-collagenous cartilage protein that is essential for the structural integrity of the cartilage extracellular matrix. The repeated modular structure of COMP allows it to “bridge” and assemble multiple cartilage extracellular matrix components such as collagens, matrilins, and proteoglycans. With its modular structure, COMP also has the potential to act as a scaffold for growth factors, thereby affecting how and when the growth factors are presented to cell-surface receptors. However, it is not known whether COMP binds growth factors. We studied the binding interaction between COMP and TGF-β1 in vitro and determined the effect of COMP on TGF-β1-induced signal transduction in reporter cell lines and primary cells. Our results demonstrate that mature COMP protein binds to multiple TGF-β1 molecules and that the peak binding occurs at slightly acidic pH. These interactions were confirmed by dual polarization interferometry and visualized by rotary shadow electron microscopy. There is cation-independent binding of TGF-β1 to the C-terminal domain of COMP. In the presence of manganese, an additional TGF-β-binding site is present in the TSP3 repeats of COMP. Finally, we show that COMP-bound TGF-β1 causes increased TGF-β1-dependent transcription. We conclude that TGF-β1 binds to COMP and that TGF-β1 bound to COMP has enhanced bioactivity.