Julia T. Oxford

Calpain-Cleavage of α-Synuclein: Connecting Proteolytic Processing to Disease-Linked Aggregation

Brian M. Dufty et al. — Tue, 13 Dec 2011 12:21:02 PST

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are both characterized pathologically by the presence of neuronal inclusions termed Lewy bodies (LBs). A common feature found in LBs are aggregates of α-synuclein (α-Syn), and although it is now recognized that α-Syn is the major building block for these toxic filaments, the mechanism of how this occurs remains unknown. In the present study, we demonstrate that proteolytic processing of α-Syn by the protease calpain I leads to the formation of aggregated high-molecular weight species and adoption of a β-sheet structure. To determine whether calpain-cleavage of α-Syn occurs in PD and DLB, we designed site-directed calpain-cleavage antibodies to α-Syn and tested their utility in several animal model systems. Detection of calpain-cleaved α-Syn was evident in mouse models of cerebral ischemia and PD and in a Drosophila model of PD. In the human PD and DLB brain, calpain-cleaved α-Syn antibodies immunolabeled LBs and neurites in the substantia nigra. Moreover, calpain-cleaved α-Syn fragments identified within LBs colocalized with activated calpain in neurons of the PD and DLB brains. These findings suggest that calpain I may participate in the disease-linked aggregation of α-Syn in various α-synucleinopathies.

Suppression of MeCP2β Expression Inhibits Neurite Extension in PC12 Cells

Sorcha M. Cusack et al. — Tue, 13 Dec 2011 12:20:57 PST

Regulation of gene expression is critical to the proper development of neuronal cells. The methyl-CpG binding protein 2 (MeCP2) operates as a transcriptional repressor by facilitating histone deacetylation and DNA methylation-dependent transcriptional silencing. This study examined the importance of MeCP2 in the regulation of neurite formation in PC12 cells. Expression of MeCP2 increased in a time-dependent manner after induction of neuronal differentiation. Expression was assessed at both the transcriptional and translation levels, and reached a maximum at 24 h post-induction. In addition, a marked inhibition of neurite extension and proper localization of a marker for synapse formation, synapsin I, were observed when MeCP2 expression was decreased by the addition of an antisense morpholino oligomer directed to the translational initiation site for MeCP2β. The removal of the antisense oligomer allowed neurite extension to progress. However, the addition of antisense oligomer to previously differentiated PC12 cells did not affect established neurite processes. Taken collectively, our results indicate a role for MeCP2β early in the events of neurite formation and that the relative levels of MeCP2α and MeCP2β may be different in early differentiating neurons than is found in the adult brain. In addition, unique functions may exist for the two isoforms of MeCP2. Our results indicate that the inhibition of neurite elaboration caused by a reduction in MeCP2 may be reversible.

Collagen a1(XI) in Normal and Malignant Breast Tissue

Karen C. Halsted et al. — Tue, 13 Dec 2011 12:20:51 PST

Little is known about collagen XI expression in normal and malignant breast tissue. Tissue microarrays, constructed from 72 patients with breast carcinoma and matched normal tissue, were immunohistochemically stained with five antisera against isoform-specific regions of collagen a

1(XI) N-terminal domain. Staining intensity was graded on a 0–3 scale in epithelial cytoplasm, stroma, and endothelial staining of the vasculature of each tissue core. The staining was compared to known pathologic parameters: age, tumor size, overall tumor grade, nuclear grade, tubule formation, mitotic counts, angiolymphatic invasion, node status, estrogen receptor status, progesterone receptor status, and HER-2/neu status. Estrogen and progesterone receptor status were used as a control for comparison. With antisera V1a and amino propeptide (Npp), stroma surrounding cancerous cells was found to have decreased collagen a 1(XI) staining compared to stroma adjacent to normal epithelium (P¼0.0006, Po0.0001). Collagen a 1(XI) staining with V1a antiserum in cytoplasm of cancer cells demonstrated decreased intensity in metastasized primary tumors when compared to nonmetastasized primary tumors (P¼0.009). Cytoplasmic staining with Npp antiserum in cancer demonstrated an inverse relationship to positive estrogen receptor status in cancer (P¼0.012) and to progesterone receptor status (P¼0.044). Stromal staining for Npp in cancerous tissue demonstrated an inverse relationship with tubule formation score (P¼0.015). This is the first study to localize collagen XI within normal and malignant breast tissue. Collagen a

1(XI) appears to be downregulated in stroma surrounding breast cancer. Detection of collagen XI in breast tissue may help predict women who have lymph node metastases.

Clinical Significance of Interleukin (IL)-6 in Cancer Metastasis to Bone: Potential of Anti-IL-6 Therapies

Ken Tawara et al. — Tue, 13 Dec 2011 12:20:46 PST

Metastatic events to the bone occur frequently in numerous cancer types such as breast, prostate, lung, and renal carcinomas, melanoma, neuroblastoma, and multiple myeloma. Accumulating evidence suggests that the inflammatory cytokine interleukin (IL)-6 is frequently upregulated and is implicated in the ability of cancer cells to metastasize to bone. IL-6 is able to activate various cell signaling cascades that include the STAT (signal transducer and activator of transcription) pathway, the PI3K (phosphatidylinositol-3 kinase) pathway, and the MAPK (mitogen-activated protein kinase) pathway. Activation of these pathways may explain the ability of IL-6 to mediate various aspects of normal and pathogenic bone remodeling, inflammation, cell survival, proliferation, and pro-tumorigenic effects. This review article will discuss the role of IL-6: 1) in bone metabolism, 2) in cancer metastasis to bone, 3) in cancer prognosis, and 4) as potential therapies for metastatic bone cancer.

Proteomic Analysis of Col11a1-associated Protein Complexes

Raquel Brown et al. — Tue, 13 Dec 2011 12:20:41 PST

Cartilage plays an essential role during skeletal development within the growth plate and in articular joint function. Interactions between the collagen fibrils and other extracellular matrix molecules maintain structural integrity of cartilage, orchestrate complex dynamic events during embryonic development, and help to regulate fibrillogenesis. To increase our understanding of these events, affinity chromatography and liquid chromatography/tandem mass spectrometry were used to identify proteins that interact with the collagen fibril surface via the amino terminal domain of collagen alpha 1(XI) a protein domain that is displayed at the surface of heterotypic collagen fibrils of cartilage. Proteins extracted from fetal bovine cartilage using homogenization in high ionic strength buffer were selected based on affinity for the amino terminal noncollagenous domain of collagen alpha 1(XI). Mass spectrometry was used to determine the amino acid sequence of tryptic fragments for protein identification. Extracellular matrix molecules and cellular proteins that were identified as interacting with the amino terminal domain of collagen alpha 1(XI) directly or indirectly, included proteoglycans, collagens, and matricellular molecules, some of which also play a role in fibrillogenesis, while others are known to function in the maintenance of tissue integrity. Characterization of these molecular interactions will provide a more thorough understanding of how the extracellular matrix molecules of cartilage interact and what role collagen XI plays in the process of fibrillogenesis and maintenance of tissue integrity. Such information will aid tissue engineering and cartilage regeneration efforts to treat cartilage tissue damage and degeneration.

Immunohistochemical Localization of Collagen Type XI α1 and α2 Chains in Human Colon Tissue

Karen B. Bowen et al. — Tue, 13 Dec 2011 12:20:38 PST

In previous studies, collagen XI mRNA has been detected in colon cancer, but its location in human colon tissue has not been determined. The heterotrimeric collagen XI consists of three a chains. While it is known that collagen XI plays a regulatory role in collagen fibril formation, its function in the colon is unknown. The characterization of normal human colon tissue will allow a better understanding of the variance of collagen XI in abnormal tissues. Grossly normal and malignant human colon tissue was obtained from pathology archives. Immunohistochemical staining with a 58K Golgi marker and α1(XI) and α2(XI) antisera was used to specifically locate their presence in normal colon tissue. A comparative bright field microscopic analysis showed the presence of collagen XI in human colon. The juxtanuclear, dot-like collagen XI staining in the Golgi apparatus of goblet cells in normal tissue paralleled the staining of the 58K Golgi marker. Ultra light microscopy verified these results. Staining was also confirmed in malignant colon tissue. This study is the first to show that collagen XI is present in the Golgi apparatus of normal human colon goblet cells and localizes collagen XI in both normal and malignant tissue. Although the function of collagen XI in the colon is unknown, our immunohistochemical characterization provides the foundation for future immunohistopathology studies of the colon.

Caspase Activation Independent of Cell Death is Required for Proper Cell Dispersal and Correct Morphology in PC12 Cells

Troy T. Rohn et al. — Tue, 13 Dec 2011 12:20:33 PST

Caspase activation is indispensable for the proper execution of apoptosis. However, to date, little is known about other possible physiologic functions for this class of enzymes in addition to their well-defined role in apoptosis. In this report, we described an action of caspase-3 involving cell dispersion that is independent of cell death. Using an in vitro neuronal model system consisting of PC12 cells, we observed a transient activation of caspase-3 both in situ and by Western blot analysis that was evident at 1 h following plating, was maximal by 3 h, and was attenuated by 24 h. Preincubation of PC12 cells with either the caspase-3 inhibitor, DEVD, or antisense caspase-3 oligonucleotides caused cells to be more rounded in appearance and led to a failure of cells to disperse properly. Additional experiments demonstrated a possible target for caspase cleavage to be the cytoskeletal protein, tau. These data suggest a requirement for caspase activation and subsequent disassembly of the cytoskeleton during cell dispersion and represent a novel role for caspases that may allow for proper migration of neurons to target locations during development.

Deamidation Destabilizes and Triggers Aggregation of a Lens Protein, βA3-crystallin

Takumi Takata et al. — Tue, 13 Dec 2011 12:20:29 PST

Protein aggregation is a hallmark of several neurodegenerative diseases and also of cataracts. The major proteins in the lens of the eye are crystallins, which accumulate throughout life and are extensively modified. Deamidation is the major modification in the lens during aging and cataracts. Among the crystallins, the βA3-subunit has been found to have multiple sites of deamidation associated with the insoluble proteins in vivo. Several sites were predicted to be exposed on the surface of βA3 and were investigated in this study. Deamidation was mimicked by site-directed mutagenesis at Q42 and N54 on the N-terminal domain, N133 and N155 on the C-terminal domain, and N120 in the peptide connecting the domains. Deamidation altered the tertiary structure without disrupting the secondary structure or the dimer formation of βA3. Deamidations in the C-terminal domain and in the connecting peptide decreased stability to a greater extent than deamidations in the N-terminal domain. Deamidation at N54 and N155 also disrupted the association with the βB1-subunit. Sedimentation velocity experiments integrated with high-resolution analysis detected soluble aggregates at 15%–20% in all deamidated proteins, but not in wild-type βA3. These aggregates had elevated frictional ratios, suggesting that they were elongated. The detection of aggregates in vitro strongly suggests that deamidation may contribute to protein aggregation in the lens. A potential mechanism may include decreased stability and/or altered interactions with other β-subunits. Understanding the role of deamidation in the long-lived crystallins has important implications in other aggregation diseases.

Characterization of Collagenous Matrix Assembly in a Chondrocyte Model System

Sorcha Yingst et al. — Fri, 08 Jul 2011 07:54:54 PDT

Collagen is a major component of the newly synthesized pericellular microenvironment of chondrocytes. Collagen types II, IX, and XI are synthesized and assembled into higher ordered complexes by a mechanism in which type XI collagen plays a role in nucleation of new fibrils, and in limiting fibril diameter. This study utilizes a cell line derived from the Swarm rat chondrosarcoma that allows the accumulation and assembly of pericellular matrix. Immunofluorescence and atomic force microscopy were used to assess early intermediates of fibril formation. Results indicate that this cell line synthesizes and secretes chondrocyte-specific pericellular matrix molecules including types II, IX, and XI collagen and is suitable for the study of newly synthesized collagen matrix under the experimental conditions used. AFM data indicate that small fibrils or assemblies of microfibrils are detectable and may represent precursors of the ∼20 nm thin fibrils reported in cartilage. Treatment with hyaluronidase indicates that the dimensions of the small fibrils may be dependent upon the presence of hyaluronan within the matrix. This study provides information on the composition and organization of the newly synthesized extracellular matrix that plays a role in establishing the material properties and performance of biological materials such as cartilage.

Immuno-SEM Characterization of Developing Bovine Cartilage

Michelle Gerritsen et al. — Thu, 09 Jun 2011 15:26:42 PDT

Collagen is a vital material in the tissues of living organisms. Found almost everywhere in the human body, collagen is important in connective tissues, bone growth, and cartilage. Collagen XI makes up a very small portion of the cartilaginous tissue; however, it plays a key role in cartilaginous tissue. Collagen XI and two collagen XI isoforms, V1b and V2, are critical in the ossification process. The location of collagen XI, V1b, V2, and their specific functions in the ossification process within developing bovine cartilage are not well characterized. In this work, the location of collagens I, II, XI and two collagen XI isoforms, V1b and V2, present in developing bovine cartilage are investigated using the immuno-SEM technique. The results for the locations of collagen I and II indicate a high level of consistency with previous work, thus showing that the technique of immuno-SEM can be used with confidence to determine the location of various collagen types within cartilaginous and mineralized tissue. This work has shown that collagen XI is present in the lower hypertrophic region and also in a pericellular arrangement, within about two microns of cell walls, throughout the cartilaginous tissue. V1b is expressed in the articular surface, mineralized region, resting zone, and the distal edge of the diaphysis. The V2 isoform is most strongly expressed in areas of newly forming cartilage, and disappears with chondrocyte maturation. V2 is present in the distal edge of the epiphysis, as well as in mineralized tissue. Collagen XI and two of its isoforms, V1b and V2, are thought to play a critical role in the ossification process. However, this role is not well understood, and is still being characterized. The detection of collagen XI and two of its isoforms in the osteo-chondral junction as well as at a joint surface further point to collagen XI, V1b, and V2 playing a vital role in the ossification process, and warrants further research as to their specific function within the ossification process.

The Expression Patterns of Minor Fibrillar Collagens During Development in Zebrafish

Ming Fang et al. — Fri, 18 Mar 2011 08:16:50 PDT

Minor fibrillar collagens are recognized as the organizers and nucleators during collagen fibrillogenesis but likely serve additional functions. The minor fibrillar collagens include collagens type V and type XI. Mutations of collagen type V and XI can cause Ehlers Danlos, Stickler's, and Marshall's syndromes in human. We have characterized the spatiotemporal expression patterns of Col11a1, Col11a2, Col5a1 as well as Col5a3 in zebrafish embryos by in situ hybridization. Col5a1 is expressed in developing somites, neural crest, the head mesenchyme, developing cranial cartilage, pharyngeal arches and vertebrae. Col5a3 is detected in the notochord, mesenchyme cells in the eyes and lens. Both Col11a1 and Col11a2 have similar expression patterns, including notochord, otic vesicle, and developing cranial cartilages. Zebrafish may therefore serve as a valuable vertebrate model system for the study of diseases associated with collagens type V and XI mutations.

Inhibition of Proprotein Convertase Ski-1 Blocks Transcription of Key Extracellular Matrix Genes Regulating Osteoblastic Mineralization

Jeff P. Gorski et al. — Wed, 16 Mar 2011 15:32:23 PDT

Mineralization, a characteristic phenotypic property of osteoblastic lineage cells, was blocked by AEBSF and dec-RRLL-cmk, inhibitors of SKI-1 (site 1; subtilisin kexin like-1) protease. Since SKI-1 is required for activation of SREBP and CREB/ATF family transcription factors, we tested the effect of these inhibitors on gene expression. AEBSF decreased expression of 140 genes by 1.5- to 3.0-fold including Phex, Dmp1, COL1A1, COL11A1 and fibronectin. Direct comparison of AEBSF and dec-RRLL-cmk, a more specific SKI-1 inhibitor, demonstrated that expression of Phex, Dmp1, COL11A1 and fibronectin was reduced by both while COL1A2 and HMGCS1 were reduced only by AEBSF. AEBSF and dec-RRLL-cmk decreased the nuclear content of SKI-1 activated forms of transcription factors SREBP-1, SREBP-2, and OASIS. In contrast to AEBSF, the actions of dec-RRLL-cmk represent the sum of its direct actions on SKI-1 and indirect actions on caspase-3. Specifically, dec-RRLL-cmk reduced intracellular caspase-3 activity by blocking the formation of activated 19 kDa caspase-3. Conversely, over-expression of SKI-1 activated SREBP-1a and CREB-H in UMR106-01 osteoblastic cells increased the number of mineralized foci and altered their morphology to yield mineralization nodules, respectively. In summary, SKI-1 regulates the activation of transmembrane transcription factor precursors required for expression of key genes required for mineralization of osteoblastic cultures in vitro and bone formation in vivo. Our results indicate that the differentiated phenotype of osteoblastic cells, and possibly osteocytes, depends upon the non-apoptotic actions of SKI-1.

Differential Expression of Collagen Type V and XI α-1 in Human Ascending Thoracic Aortic Aneurysms

Julia Oxford — Thu, 24 Feb 2011 14:05:17 PST

Background—The molecular mechanisms leading to ascending thoracic aortic aneurysms (ATAAs) remain unknown. We hypothesized that alterations in expression levels of specific fibrillar collagens occur during the aneurysmal process.

Methods—Surgical samples from ascending aortas from patients with degenerative ATAAs were subdivided by aneurysm diameter: small, 5 to 6 cm; medium, 6 to 7 cm; and large, greater than 7 cm; and compared with nonaneurysmal aortas (mean diameter, 2.3 cm).

Results—Histology, immunofluorescence, and electron microscopy demonstrated greater disorganization of extracellular matrix constituents in ATAAs as compared with control with an increase in collagen α1(XI) within regions of cystic medial degenerative lesions. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed collagens type V and α1(XI) were significantly and linearly increased in ATAAs as compared with control (p < 0.001). There was no change in the messenger ribonucleic acid (mRNA) expression levels of collagens type I and III. Western blot analysis showed collagens type I and III were significantly decreased and collagens α1(XI) and V were significantly increased and were linearly correlated with the size of the aneurysm (p < 0.001 for both).

Conclusions—These results demonstrate that increased collagen α1(XI) and collagen V mRNA and protein levels are linearly correlated with the size of the aneurysm and provide a potential mechanism for the generation and progression of aneurysmal enlargement.

Collagen 11a1 is Indirectly Activated by Lymphocyte Enhancer-binding Factor 1 (Lef1) and Negatively Regulates Osteoblast Maturation

Rachel A. Kahler et al. — Tue, 08 Feb 2011 15:30:38 PST

Alpha 1 (XI) collagen (Col11a1) is essential for normal skeletal development. Mutations in Col11a1 cause Marshall and Stickler syndromes, characterized by craniofacial abnormalities, nearsightedness and hearing abnormalities. Despite its link to human diseases, few studies have characterized the factors that control Col11a1 transcription. We previously identified Col11a1 as a differentially expressed gene in Lef1-suppressed MC3T3 preosteoblasts. Here we report that Lef1 activates the Col11a1 promoter. This activation is dependent upon the DNA binding domain of Lef1, but does not require the ß-catenin interaction domain, suggesting that it is not responsive to Wnt signals. Targeted deletion of Col11a1 with an antisense morpholino accelerated osteoblastic differentiation and mineralization in C2C12 cells, similar to what was observed in Lef1-suppressed MC3T3 cells. Moreover incubation with a purified Col11a1 N-terminal fragment, V1B, prevented alkaline phosphatase expression in MC3T3 and C2C12 cells. These results suggest that Lef1 is an activator of the Col11a1 promoter and that Col11a1 suppresses terminal osteoblast differentiation.

Studies of Type XI Collagen Interaction

K. Irwin et al. — Tue, 08 Feb 2011 15:30:37 PST

Bone Microarchitecture Is Dependent Upon Collagen α1(XI) Expression During Development

Julia T. Oxford — Thu, 23 Apr 2009 18:21:47 PDT

Collagen type XI is an essential component of the collagen fibrils within the developing skeleton. It is a quantitatively minor member of the fibrillar collagens that plays a regulatory role in the assembly of embryonic collagen fibrils as the diameter of collagen fibrils is dependent on the presence of Collagen α1(XI). However, very little is known about the role of collagen type XI in bone microarchitecture formation. In this study, skeletal mineralization was evaluated in the absence and presence of Collagen α1(XI) comparing the chondrodysplasia (cho) mouse embryo to wildtype. The data presented support a role for collagen type α1(XI) in skeletal development that is distinct from its function in nucleating the formation and limiting the diameter of cartilage type II collagen fibrils. Established characteristics of the cho mouse were apparent in the mouse included in this study, including a shortened snout, and short, wide long-bones with flared metaphyses. The cho mouse humerus was 55% the length of the wildtype humerus and approximately 35% wider at the diaphysis and metaphyses. Quantification of the differences in the humerus, vertebral column, and ribcage of the cho and wildtype mice was carried out using high-resolution three-dimensional models that were created from x-ray microcomputed tomography images. Analysis revealed differences in bone density, size and microarchitecture. The differences in skeletal properties between these mice were analyzed by determining bone mineral density (BMD) calculations and three-dimensional measures of microarchitecture (BV/TV, Tb.Th, Tb.N, Tb.Sp). Results indicated that bone mineral density was increased in the absence of collagen α1(XI) in humerus, vertebrae, and the anterior rib samples of the cho mouse. The increase in bone density correlated with an observed increase in trabecular number, trabecular thickness, and percent bone volume, as well as a decrease in trabecular separation in the humerus, ribs, and vertebrae. In contrast, cortical bone mineral density was similar in wildtype and cho mice. A function for Collagen α1(XI) in the establishment of bone microarchitecture during embryonic development is supported by this data. Future studies will focus on Collagen α1(XI) in bone formation and mineralization by osteoblasts, and new functions for Collagen α1(XI) in non-cartilaginous tissues of the skeleton.

Characterization of Developing Bovine Cartilage Using Immuno-SEM

Michelle Gerritsen et al. — Mon, 13 Apr 2009 13:50:10 PDT

Collagen is an important material in tissues of living organisms. Found almost everywhere in the human body, it is important in connective tissues, bone growth, and cartilage. In this work, collagen XI and two of its isoforms, V1b and V2, present in developing cartilage, are investigated using the technique of immuno-SEM. The efficacy of this technique to examine fundamental issues related to ossification is presented. Prior work using alternative techniques has shown that both isoforms were observed in the longitudinal septa, and in a very restricted pericellular pattern in the resting zone. Prior to primary ossification, V1b was detected only in the diaphysis, primarily adjacent to the perichondrium, and not in the epiphysis. The V2-containing isoforms were most strongly expressed in areas of newly forming cartilage, and disappeared as chondrocyte maturation proceeded. The technique of immuno-SEM will yield a better understanding of protein composition and organization within the osteochondral junction.

Interaction between Amino Propeptides of Type XI Procollagen 1 Chains

Julia T. Oxford — Thu, 15 Jan 2009 21:26:40 PST

Type XI collagen is a quantitatively minor yet essential constituent of the cartilage extracellular matrix. The amino propeptide of the 1 chain remains attached to the rest of the molecule for a longer period of time after synthesis than the other amino propeptides of type XI collagen and has been localized to the surface of thin collagen fibrils. Yeast two-hybrid system was used to demonstrate that a homodimer of 1(XI) amino propeptide (1(XI)Npp) could form in vivo. Interaction was also confirmed using multi-angle laser light scattering, detecting an absolute weight average molar mass ranging from the size of a monomer to the size of a dimer (25,000–50,000 g/mol), respectively. Binding was shown to be saturable by ELISA. An interaction between recombinant 1(XI)Npp and the endogenous 1(XI)Npp was observed, and specificity for 1(XI)Npp but not 2(XI)Npp was demonstrated by co-precipitation. The interaction between the recombinant form of 1(XI)Npp and the endogenous 1(XI)Npp resulted in a stable association during the regeneration of cartilage extracellular matrix by fetal bovine chondrocytes maintained in pellet culture, generating a protein that migrated with an apparent molecular mass of 50–60 kDa on an SDS-polyacrylamide gel.