Differentiated activities of decorin and biglycan in the progression of post-traumatic osteoarthritis.

OBJECTIVE: To delineate the activities of decorin and biglycan in the progression of post-traumatic osteoarthritis (PTOA). DESIGN: Three-month-old inducible biglycan (BgniKO) and decorin/biglycan compound (Dcn/BgniKO) knockout mice were subjected to the destabilization of the medial meniscus (DMM) surgery to induce PTOA. The OA phenotype was evaluated by assessing joint structure and sulfated glycosaminoglycan (sGAG) staining via histology, surface collagen fibril nanostructure and calcium content via scanning electron microscopy, tissue modulus via atomic force microscopy-nanoindentation, as well as subchondral bone structure and meniscus ossification via micro-computed tomography. Outcomes were compared with previous findings in the inducible decorin (DcniKO) knockout mice. RESULTS: In the DMM model, BgniKO mice developed similar degree of OA as the control (0.44 [-0.18 1.05] difference in modified Mankin score), different from the more severe OA phenotype observed in DcniKO mice (1.38 [0.91 1.85] difference). Dcn/BgniKO mice exhibited similar histological OA phenotype as DcniKO mice (1.51 [0.97 2.04] difference vs control), including aggravated loss of sGAGs, salient surface fibrillation and formation of osteophyte. Meanwhile, Dcn/BgniKO mice showed further cartilage thinning than DcniKO mice, resulting in the exposure of underlying calcified tissues and aberrantly high surface modulus. BgniKO and Dcn/BgniKO mice developed altered subchondral trabecular bone structure in both Sham and DMM groups, while DcniKO and control mice did not. CONCLUSION: In PTOA, decorin plays a more crucial role than biglycan in regulating cartilage degeneration, while biglycan is more important in regulating subchondral bone structure. The two have distinct activities and modest synergy in the pathogenesis of PTOA.

Decorin is an autophagy-inducible proteoglycan and is required for proper in vivo autophagy.

We have recently discovered that soluble extracellular matrix constituents regulate autophagy via an outside-in signaling pathway. Decorin, a secreted proteoglycan, evokes autophagy in endothelial cells and mitophagy in breast carcinoma cells. However, it is not known whether decorin expression can be regulated by autophagic stimuli such as mTOR inhibition or nutrient deprivation. Thus, we tested whether pro-autophagic stimuli could affect decorin expression in mouse cardiac tissue and whether the absence of decorin could disrupt the in vivo autophagic response. We found that nutrient deprivation induced decorin at the mRNA and protein level in vivo and in vitro, a process regulated at the transcriptional level by inhibiting the canonical mTOR pathway. Moreover, Dcn-/- mice displayed an aberrant response to fasting compared to wild-type mice. Our study establishes a new role for an extracellular matrix proteoglycan and provides a mechanistic role for soluble decorin in regulating a fundamental intracellular catabolic process.

Achilles tendons from decorin- and biglycan-null mouse models have inferior mechanical and structural properties predicted by an image-based empirical damage model.

Achilles tendons are a common source of pain and injury, and their pathology may originate from aberrant structure function relationships. Small leucine rich proteoglycans (SLRPs) influence mechanical and structural properties in a tendon-specific manner. However, their roles in the Achilles tendon have not been defined. The objective of this study was to evaluate the mechanical and structural differences observed in mouse Achilles tendons lacking class I SLRPs; either decorin or biglycan. In addition, empirical modeling techniques based on mechanical and image-based measures were employed. Achilles tendons from decorin-null (Dcn(-/-)) and biglycan-null (Bgn(-/-)) C57BL/6 female mice (N=102) were used. Each tendon underwent a dynamic mechanical testing protocol including simultaneous polarized light image capture to evaluate both structural and mechanical properties of each Achilles tendon. An empirical damage model was adapted for application to genetic variation and for use with image based structural properties to predict tendon dynamic mechanical properties. We found that Achilles tendons lacking decorin and biglycan had inferior mechanical and structural properties that were age dependent; and that simple empirical models, based on previously described damage models, were predictive of Achilles tendon dynamic modulus in both decorin- and biglycan-null mice.

A decorin-deficient matrix affects skin chondroitin/dermatan sulfate levels and keratinocyte function.

Decorin is a small leucine-rich proteoglycan harboring a single glycosaminoglycan chain, which, in skin, is mainly composed of dermatan sulfate (DS). Mutant mice with targeted disruption of the decorin gene (Dcn(-/-)) exhibit an abnormal collagen architecture in the dermis and reduced tensile strength, collectively leading to a skin fragility phenotype. Notably, Ehlers-Danlos patients with mutations in enzymes involved in the biosynthesis of DS display a similar phenotype, and recent studies indicate that DS is involved in growth factor binding and signaling. To determine the impact of the loss of DS-decorin in the dermis, we analyzed the glycosaminoglycan content of Dcn(-/-) and wild-type mouse skin. The total amount of chondroitin/dermatan sulfate (CS/DS) was increased in the Dcn(-/-) skin, but was overall less sulfated with a significant reduction in bisulfated ΔDiS2,X (X=4 or 6) disaccharide units, due to the reduced expression of uronyl 2-O sulfotransferase (Ust). With increasing age, sulfation declined; however, Dcn(-/-) CS/DS was constantly undersulfated vis-à-vis wild-type. Functionally, we found altered fibroblast growth factor (Fgf)-7 and -2 binding due to changes in the micro-heterogeneity of skin Dcn(-/-) CS/DS. To better delineate the role of decorin, we used a 3D Dcn(-/-) fibroblast cell culture model. We found that the CS/DS extracts of wild-type and Dcn(-/-) fibroblasts were similar to the skin sugars, and this correlated with the lack of uronyl 2-O sulfotransferase in the Dcn(-/-) fibroblasts. Moreover, Ffg7 binding to total CS/DS was attenuated in the Dcn(-/-) samples. Surprisingly, wild-type CS/DS significantly reduced the binding of Fgf7 to keratinocytes in a concentration dependent manner unlike the Dcn(-/-) CS/DS that only affected the binding at higher concentrations. Although binding to cell-surfaces was quite similar at higher concentrations, keratinocyte proliferation was differentially affected. Higher concentration of Dcn(-/-) CS/DS induced proliferation in contrast to wild-type CS/DS. 3D co-cultures of fibroblasts and keratinocytes showed that, unlike Dcn(-/-) CS/DS, wild-type CS/DS promoted differentiation of keratinocytes. Collectively, our results provide novel mechanistic explanations for the reported defects in wound healing in Dcn(-/-) mice and possibly Ehlers-Danlos patients. Moreover, the lack of decorin-derived DS and an altered CS/DS composition differentially influence keratinocyte behavior.

Decorin deficiency promotes hepatic carcinogenesis.

Hepatocellular carcinoma represents one of the most-rapidly spreading cancers in the world. In the majority of cases, an inflammation-driven fibrosis or cirrhosis precedes the development of the tumor. During malignant transformation, the tumor microenvironment undergoes qualitative and quantitative changes that modulate the behavior of the malignant cells. A key constituent for the hepatic microenvironment is the small leucine-rich proteoglycan decorin, known to interfere with cellular events of tumorigenesis mainly by blocking various receptor tyrosine kinases (RTK) such as EGFR, Met, IGF-IR, PDGFR and VEGFR2. In this study, we characterized cell signaling events evoked by decorin deficiency in two experimental models of hepatocarcinogenesis using thioacetamide or diethyl nitrosamine as carcinogens. Genetic ablation of decorin led to enhanced tumor occurrence as compared to wild-type animals. These findings correlated with decreased levels of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and a concurrent elevation in retinoblastoma protein phosphorylation via cyclin dependent kinase 4. Decreased steady state p21(Waf1/Cip1) levels correlated with enhanced expression of transcription factor AP4, a known transcriptional repressor of p21(Waf1/Cip1), and enhanced c-Myc protein levels. In addition, translocation of ß-catenin was a typical event in diethyl nitrosamine-evoked tumors. In parallel, decreased phosphorylation of both c-Myc and ß-catenin was observed in Dcn(-/-) livers likely due to the hindered GSK3ß-mediated targeting of these proteins to proteasomal degradation. We discovered that in a genetic background lacking decorin, four RTKs were constitutively activated (phosphorylated), including three known targets of decorin such as PDGFRα, EGFR, IGF-IR, and a novel RTK MSPR/RON. Our findings provide powerful genetic evidence for a crucial in vivo role of decorin during hepatocarcinogenesis as lack of decorin in the liver and hepatic stroma facilitates experimental carcinogenesis by providing an environment devoid of this potent pan-RTK inhibitor. Thus, our results support future utilization of decorin as an antitumor agent in liver cancer.

Lack of decorin expression by human bladder cancer cells offers new tools in the therapy of urothelial malignancies.

Decorin, a multifunctional small leucine-rich extracellular matrix proteoglycan, has been shown to possess potent antitumour activity. However, there is some uncertainty whether different cancer cells express decorin in addition to non-malignant stromal cells. In this study we clarified decorin expression by human bladder cancer cells both in vivo and in vitro. In addition, the effect of adenovirus-mediated decorin expression on human bladder cancer cells in vitro was examined. We first demonstrated using the publicly available GeneSapiens databank that decorin gene expression is present in both normal and malignant human bladder tissues. However, when we applied in situ hybridization with digoxigenin-labeled RNA probes for decorin on human bladder carcinoma tissue samples derived from a large radical cystectomy patient cohort (n = 199), we unambiguously demonstrated that invasive and non-invasive bladder carcinoma cells completely lack decorin mRNA. The cancer cells were also negative for decorin immunoreactivity. Instead, decorin expression was localized solely to original non-malignant stromal areas of bladder tissue. In accordance with the aforementioned results, human bladder cancer cells in vitro were also negative for decorin expression as shown by RT-qPCR analyses. The lack of decorin expression by bladder cancer cells was shown not to be due to the methylation of the proximal promoter region of the decorin gene. When bladder cancer cells were transfected with a decorin adenoviral vector, their proliferation was significantly decreased. In conclusion, we have shown that human bladder cancer cells are totally devoid of decorin expression. We have also shown that adenovirus-mediated decorin gene transduction of human bladder cancer cell lines markedly inhibits their proliferation. Thus, decorin gene delivery offers new potential therapeutic tools in urothelial malignancies.

Effect of age and proteoglycan deficiency on collagen fiber re-alignment and mechanical properties in mouse supraspinatus tendon.

Collagen fiber realignment is one mechanism by which tendon responds to load. Re-alignment is altered when the structure of tendon is altered, such as in the natural process of aging or with alterations of matrix proteins, such as proteoglycan expression. While changes in re-alignment and mechanical properties have been investigated recently during development, they have not been studied in (1) aged tendons, or (2) in the absence of key proteoglycans. Collagen fiber re-alignment and the corresponding mechanical properties are quantified throughout tensile mechanical testing in both the insertion site and the midsubstance of mouse supraspinatus tendons in wild type (WT), decorin-null (Dcn(-/-)), and biglycan-null (Bgn(-/-)) mice at three different ages (90 days, 300 days, and 570 days). Percent relaxation was significantly decreased with age in the WT and Dcn(-/-) tendons, but not in the Bgn(-/-) tendons. Changes with age were found in the linear modulus at the insertion site where the 300 day group was greater than the 90 day and 570 day group in the Bgn(-/-) tendons and the 90 day group was smaller than the 300 day and 570 day groups in the Dcn(-/-) tendons. However, no changes in modulus were found across age in WT tendons were found. The midsubstance fibers of the WT and Bgn(-/-) tendons were initially less aligned with increasing age. The re-alignment was significantly altered with age in the WT tendons, with older groups responding to load later in the mechanical test. This was also seen in the Dcn(-/-) midsubstance and the Bgn(-/-) insertion, but not in the other locations. Although some studies have found changes in the WT mechanical properties with age, this study did not support those findings. However, it did show fiber re-alignment changes at both locations with age, suggesting a breakdown of tendon's ability to respond to load in later ages. In the proteoglycan-null tendons however, there were changes in the mechanical properties, accompanied only by location-dependent re-alignment changes, suggesting a site-specific role for these molecules in loading. Finally, changes in the mechanical properties did not occur in concert with changes in re-alignment, suggesting that typical mechanical property measurements alone are insufficient to describe how structural alterations affect tendon's response to load.

The dermatan sulfate proteoglycan decorin modulates α2ß1 integrin and the vimentin intermediate filament system during collagen synthesis.

Decorin, a small leucine-rich proteoglycan harboring a dermatan sulfate chain at its N-terminus, is involved in regulating matrix organization and cell signaling. Loss of the dermatan sulfate of decorin leads to an Ehlers-Danlos syndrome characterized by delayed wound healing. Decorin-null (Dcn(-/-)) mice display a phenotype similar to that of EDS patients. The fibrillar collagen phenotype of Dcn(-/-) mice could be rescued in vitro by decorin but not with decorin lacking the glycosaminoglycan chain. We utilized a 3D cell culture model to investigate the impact of the altered extracellular matrix on Dcn(-/-) fibroblasts. Using 2D gel electrophoresis followed by mass spectrometry, we identified vimentin as one of the proteins that was differentially upregulated by the presence of decorin. We discovered that a decorin-deficient matrix leads to abnormal nuclear morphology in the Dcn(-/-) fibroblasts. This phenotype could be rescued by the decorin proteoglycan but less efficiently by the decorin protein core. Decorin treatment led to a significant reduction of the α2ß1 integrin at day 6 in Dcn(-/-) fibroblasts, whereas the protein core had no effect on ß1. Interestingly, only the decorin core induced mRNA synthesis, phosphorylation and de novo synthesis of vimentin indicating that the proteoglycan decorin in the extracellular matrix stabilizes the vimentin intermediate filament system. We could support these results in vivo, because the dermis of wild-type mice have more vimentin and less ß1 integrin compared to Dcn(-/-). Furthermore, the α2ß1 null fibroblasts also showed a reduced amount of vimentin compared to wild-type. These data show for the first time that decorin has an impact on the biology of α2ß1 integrin and the vimentin intermediate filament system. Moreover, our findings provide a mechanistic explanation for the reported defects in wound healing associated with the Dcn(-/-) phenotype.

Influence of decorin on the mechanical, compositional, and structural properties of the mouse patellar tendon.

The interactions of small leucine-rich proteoglycans (SLRPs) with collagen fibrils, their association with water, and their role in fibrillogenesis suggests that SLRPs may play an important role in tendon mechanics. Some studies have assessed the role of SLRPs in the mechanical response of the tendon, but the relationships between sophisticated mechanics, assembly of collagen, and SLRPs have not been well characterized. Decorin content was varied in a dose dependent manner using decorin null, decorin heterozygote, and wild type mice. Quantitative measures of mechanical (tension and compression), compositional, and structural changes of the mouse patellar tendon were evaluated. Viscoelastic, tensile dynamic modulus was increased in the decorin heterozygous tendons compared to wild type. These tendons also had a significant decrease in total collagen and no structural changes compared to wild type. Decorin null tendons did not have any mechanical changes; however, a significant decrease in the average fibril diameter was found. No differences were seen between genotypes in elastic or compressive properties, and all tendons demonstrated viscoelastic mechanical dependence on strain rate and frequency. These results suggest that decorin, a member of the SLRP family, plays a role in tendon viscoelasticity that cannot be completely explained by its role in collagen fibrillogenesis. In addition, reductions in decorin do not cause large changes in indentation compressive properties, suggesting that other factors contribute to these properties. Understanding these relationships may ultimately help guide development of tissue engineered constructs or treatment modalities.

Knockdown of aberrantly expressed nuclear localized decorin attenuates tumour angiogenesis related mediators in oral cancer progression model in vitro.

BACKGROUND: Oral cancer accounts for roughly 3% of cancer cases in the world with about 350,000 newly reported cases annually and a 5-year survival rate of only 50%. Majority of oral cancers are squamous cell carcinomas that originate in the oral mucosal epithelial linings. We have previously shown that in human malignant squamous cells carcinoma (SCC-25) as well as in dysplastic oral keratinocytes (DOK), a small leucine-rich multifunctional proteoglycan decorin is aberrantly expressed and localized in the nucleus where it interacts with nuclear epidermal growth factor receptor (EGFR). Post-transcriptional silencing of nuclear decorin significantly reduced IL-8 and IL8-dependent migration and invasion in these dysplastic and malignant oral epithelia. The objective of this study was to further examine the effects of nuclear decorin silencing on angiogenesis and angiogenesis related mediators in this oral cancer progression cell line model. METHODS: We have used multiplex PCR, western blotting, and in vitro endothelial tube formation assay to study angiogenesis and related pathways in nuclear decorin silenced (stable knockdown) DOK and SCC-25 cells. RESULTS: Nuclear decorin knockdown resulted in significant down regulation of IL-8 expression, however IL-10, and TGF-ß expression was not affected in either DOK or SCC25 cells as measured by multiplex RT PCR. IL-8 receptor CXCR 1 and 2 expression was slightly lower in nuclear decorin silenced cells indicating a contributing mechanism in previously shown reduced IL-8 mediated migration and invasion phenotype in these cells. IL-8 is known to induce Matrix metalloproteinase 9 (MMP9) which not only plays a role in tumour migration and invasion but also induces angiogenic switch. We found MMP9 to be significantly reduced in nuclear decorin silenced dysplastic and malignant oral epithelia. Other potent angiogenic mediators, VEGF189 and ANG-1 were either significantly reduced or completely abrogated in these cells. Angiogenesis as measured by endothelial tube-like formations of HUVEC cells was reduced by almost 50 percent when HUVECs were incubated in the presence of conditioned medium form nuclear decorin silenced dysplastic and malignant cell lines as compared to respective controls. CONCLUSIONS: Together these results indicate that aberrantly expressed nuclear localized decorin strongly influences angiogenic potential of dysplastic and malignant oral epithelial cells.

Uterine dysfunction in biglycan and decorin deficient mice leads to dystocia during parturition.

Cesarean birth rates are rising. Uterine dysfunction, the exact mechanism of which is unknown, is a common indication for Cesarean delivery. Biglycan and decorin are two small leucine-rich proteoglycans expressed in the extracellular matrix of reproductive tissues and muscle. Mice deficient in biglycan display a mild muscular dystrophy, and, along with mice deficient in decorin, are models of Ehlers-Danlos Syndrome, a connective tissue anomaly associated with uterine rupture. As a variant of Ehlers-Danlos Syndrome is caused by a genetic mutation resulting in abnormal biglycan and decorin secretion, we hypothesized that biglycan and decorin play a role in uterine function. Thus, we assessed wild-type, biglycan, decorin and double knockout pregnancies for timing of birth and uterine function. Uteri were harvested at embryonic days 12, 15 and 18. Nonpregnant uterine samples of the same genotypes were assessed for tissue failure rate and spontaneous and oxytocin-induced contractility. We discovered that biglycan/decorin mixed double-knockout dams displayed dystocia, were at increased risk of delayed labor onset, and showed increased tissue failure in a predominantly decorin-dependent manner. In vitro spontaneous uterine contractile amplitude and oxytocin-induced contractile force were decreased in all biglycan and decorin knockout genotypes compared to wild-type. Notably, we found no significant compensation between biglycan and decorin using quantitative real time PCR or immunohistochemistry. We conclude that the biglycan/decorin mixed double knockout mouse is a model of dystocia and delayed labor onset. Moreover, decorin is necessary for uterine function in a dose-dependent manner, while biglycan exhibits partial compensatory mechanisms in vivo. Thus, this model is poised for use as a model for testing novel targets for preventive or therapeutic manipulation of uterine dysfunction.

A role for aberrantly expressed nuclear localized decorin in migration and invasion of dysplastic and malignant oral epithelial cells.

BACKGROUND: Oral cancer is the sixth most common malignancy worldwide with a mortality rate that is higher than many other cancers. Death usually occurs as a result of local invasion and regional lymph node metastases. Decorin is a multifunctional proteoglycan of the extracellular matrix that affects the biology of various types of cancer. Previously; we have shown that decorin is aberrantly expressed in the nucleus in human dysplastic oral keratinocytes (DOK) and malignant squamous cells carcinoma (SCC-25) and human biopsy tissues. In this study, we examined the role of nuclear decorin in oral cancer progression. MATERIALS AND METHODS: We have used a post-transcriptional gene silencing (RNA interference) approach to stably knockdown nuclear decorin gene expression in DOK and SCC-25 cells using a specific shRNA plasmid and a combination of immunological and molecular techniques to study nuclear decorin function in these oral epithelial cell lines. RESULTS: More than 80% decorin silencing/knockdown was achieved as confirmed by real time PCR and western blot analysis in both DOK and SCC-25 cells. This RNA interference-mediated knockdown of nuclear decorin expression resulted in significantly reduced invasion and migration in these cell lines as measured by Matrigel™ coated and uncoated Trans well chamber assays respectively. Decorin silencing also resulted in reduced IL-8 mRNA and proteins levels in these cell lines. Culturing decorin silenced DOK and SCC-25 cells, with recombinant human IL-8 or IL-8 containing conditioned medium from respective un-transfected cells for 24 h prior to migration and invasion experiments, resulted in the salvation of reduced migration and invasion phenotype. Furthermore, we found that nuclear localized decorin interacts with EGFR in the nuclear fractions of both DOK and SCC-25 cells. Interestingly, EGFR (trans) activation has previously been shown to be involved in IL-8 production in various epithelia. CONCLUSIONS: Taken together, our results indicate that nuclear localized decorin plays an important role in migration and invasion of oral cancer cells and thus may present as a novel potential target for the treatment of oral cancer.