kDa Fragments Research Articles

Abstract 420: MMI-0100 Inhibits Cardiac Fibrosis in a Model of Cardiac Myosin Binding Protein C Hypertrophic Cardiomyopathy

Background: Hypertrophic cardiomyopathy is considered one of the most common genetic heart disorders, occurring with a frequency of about 1 in 200, with approximately 30% of those affected showing mutations within the cardiac myosin-binding protein C (cMyBP-C) gene ( MYBPC3 ). Cardiac stress, as well as cMyBP-C mutations, can trigger production of a 40kDa truncated fragment derived from the amino terminus of cMyBP-C. Genetic expression of this 40kDa fragment in mouse cardiomyocytes (MyBP-C 40k ) leads to hypertrophic cardiomyopathy), fibrosis and heart failure. Fibrosis can occur in many cardiovascular diseases and the p38-MK2 signaling pathway has been implicated in a variety of fibrotic processes. Recent studies demonstrated that MK2 inhibition using the cell-permeant peptide inhibitor MMI-0100 is protective in the setting of acute myocardial infarction. We hypothesized that MMI-0100 might also be protective in a chronic model of fibrosis, produced as a result of cMyBP-C 40k cardiomyocyte expression. Methods and Results: Non-transgenic and MyBP-C 40k transgenic (Dtg) mice were given MMI-0100 or PBS daily for 30 weeks. In control groups, long term MMI-0100 was benign, with no measurable effects on cardiac anatomy, function, cell viability, hypertrophy or probability of survival. In the Dtg group, MMI-0100 treatment reduced cardiac fibrosis, decreased cardiac hypertrophy and prolonged survival. Conclusions: Pharmaceutical inhibition of p38-MK2 signaling via MMI-0100 treatment is beneficial in the context of fibrotic MyBPC 40k disease.

Thrombin-Mediated Degradation of Human Cardiac Troponin T.

Cardiac troponin T (cTnT) is an acknowledged biomarker of acute myocardial infarction (AMI) that is known to be prone to proteolytic degradation in serum. Such degradation is usually explained by the action of μ-calpain, although there could be other candidates for that role. In the current study, we explored the hypothesis that thrombin-mediated cTnT cleavage occurs as a result of the serum sample preparation. cTnT degradation was studied by using immunoblotting and mass spectrometry (MS) analysis. The comparison of cTnT isolated from AMI heparin plasma and serum samples showed that cTnT in the plasma samples was mainly present as the full-sized molecule (approximately 35 kDa), while in serum samples it was present as a 29-kDa fragment. The incubation of recombinant cTnT, or native ternary cardiac troponin complex with thrombin or in normal human serum (NHS), resulted in the formation of a 29-kDa product that was similar to that detected in AMI serum samples. No cTnT degradation was observed when thrombin or NHS was pretreated with hirudin, a specific inhibitor of thrombin, or during incubation of troponin in normal heparin plasma. When the products of thrombin-mediated cTnT proteolysis were analyzed by MS, 2 fragments consisting of amino acid residues (aar) 2-68 and 69-288 were identified, which suggests that thrombin cleaves cTnT between R68 and S69. The results of this study suggest that the 29-kDa fragment of cTnT in AMI serum samples mainly appears due to the cleavage by thrombin during serum sample preparation.

C-terminal fragments of the amyloid precursor protein in cerebrospinal fluid as potential biomarkers for Alzheimer disease

This study assesses whether C-terminal fragments (CTF) of the amyloid precursor protein (APP) are present in cerebrospinal fluid (CSF) and their potential as biomarkers for Alzheimer’s disease (AD). Immunoprecipitation and simultaneous assay by Western blotting using multiplex fluorescence imaging with specific antibodies against particular domains served to characterize CTFs of APP in human CSF. We demonstrate that APP-CTFs are detectable in human CSF, being the most abundant a 25-kDa fragment, probably resulting from proteolytic processing by η-secretase. The level of the 25-kDa APP-CTF was evaluated in three independent CSF sample sets of patients and controls. The CSF level of this 25-kDa CTF is higher in subjects with autosomal dominant AD linked to PSEN1 mutations, in demented Down syndrome individuals and in sporadic AD subjects compared to age-matched controls. Our data suggest that APP-CTF could be a potential diagnostic biomarker for AD.

Different aggregation states of a nuclear localization signal-tagged 25-kDa C-terminal fragment of TAR RNA/DNA-binding protein 43kDa.

The mechanism and cause of motor neuronal cell death in amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disorder, are unknown; gain of function of oligomers and aggregation of misfolded proteins, including carboxyl-terminal fragments (CTFs) of TAR RNA/DNA-binding protein 43 kDa (TDP-43), have been proposed as important causative factors in the onset of ALS. We recently reported that a nuclear localization signal (NLS)-tagged 25-kDa CTF of TDP-43 (TDP25) could decrease the cell-death proportion compared with that promoted by TDP25. Here, we show oligomeric states of NLS-TDP25 and its detailed localization property using super-resolution fluorescence microscopy, FRET, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy analysis. NLS-TDP25 efficiently formed a nucleolar cap structure via RNA binding in the presence of actinomycin D, but TDP25 did not. Although cytoplasmic inclusion bodies including TDP25 had a disordered and immobile structure, NLS-TDP25 in the nucleolus was ordered and dynamic. In the diffuse state, TDP25 formed fewer oligomers and interacted with the molecular chaperone, HSP70; however, NLS-TDP25 formed oligomers. These results suggested that NLS-tagged TDP25 can change its structure to use ordered oligomeric but nontoxic state. Moreover, the structure of ordered oligomers as well as nuclear sequestration may be important in mediating cytotoxicity in ALS pathology.

Proteolytic Cleavage of the Immunodominant Outer Membrane Protein rOmpA in Rickettsia rickettsii.

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, contains two immunodominant proteins, rOmpA and rOmpB, in the outer membrane. Both rOmpA and rOmpB are conserved throughout spotted fever group rickettsiae as members of a family of autotransporter proteins. Previously, it was demonstrated that rOmpB is proteolytically processed, with the cleavage site residing near the autotransporter domain at the carboxy-terminal end of the protein, cleaving the 168-kDa precursor into apparent 120-kDa and 32-kDa fragments. The 120- and 32-kDa fragments remain noncovalently associated on the surface of the bacterium, with implications that the 32-kDa fragment functions as the membrane anchor domain. Here we present evidence for a similar posttranslational processing of rOmpA. rOmpA is expressed as a predicted 224-kDa precursor yet is observed on SDS-PAGE as a 190-kDa protein. A small rOmpA fragment of ∼32 kDa was discovered during surface proteome analysis and identified as the carboxy-terminal end of the protein. A rabbit polyclonal antibody was generated to the autotransporter region of rOmpA and confirmed a 32-kDa fragment corresponding to the calculated mass of a proteolytically cleaved rOmpA autotransporter region. N-terminal amino acid sequencing revealed a cleavage site on the carboxy-terminal side of Ser-1958 in rOmpA. An avirulent strain of R. rickettsii Iowa deficient in rOmpB processing was also defective in the processing of rOmpA. The similarities of the cleavage sites and the failure of R. rickettsii Iowa to process either rOmpA or rOmpB suggest that a single enzyme may be responsible for both processing events.IMPORTANCE Members of the spotted fever group of rickettsiae, including R. rickettsii, the etiologic agent of Rocky Mountain spotted fever, express at least four autotransporter proteins that are protective antigens or putative virulence determinants. One member of this class of proteins, rOmpB, is proteolytically processed to a passenger domain and an autotransporter domain that remain associated on the rickettsial outer membrane. The protease responsible for this posttranslation processing remains unknown. Here we show that another autotransporter, rOmpA, is similarly processed by R. rickettsii Similarities in sequence at the cleavage site and predicted secondary protein structure suggest that all four R. rickettsii autotransporters may be processed by the same outer membrane protease.

ALS-causing cleavages of TDP-43 abolish its RRM2 structure and unlock CTD for enhanced aggregation and toxicity

Pathological TDP-43 is cleaved into various fragments. Two major groups of ∼35 and ∼25 kDa have enhanced aggregation and cytotoxicity but the underlying mechanisms remain elusive. While the ∼35-kDa fragments contain entire RRM1, RRM2 and C-terminal domain (CTD) with a middle hydrophobic segment flanked by two prion-like regions; the ∼25-kDa one cleaved at Arg208 only consists of the truncated RRM2 and CTD. Remarkably, the 25-kDa fragment was characterized to induce cell death by gain of cytotoxicity and recapitulate pathological features of TDP-43 proteinopathies. Here by NMR spectroscopy we successfully characterized residue-specific conformations and inter-domain interactions of several fragments and the results show that: 1) ALS-causing truncation at Arg208 completely eliminates the intrinsic ability of RRM2 to fold, and consequently the truncated RRM2 becomes highly disordered and prone to aggregation. 2) By disrupting inter-domain interactions upon deleting the N-terminal ubiquitin-like fold in TDP-43 (102–414), the extreme C-terminal prion-like region of CTD is released, while in TDP-43 (208–414), almost the whole CTD is unlocked. As CTD itself is prone to aggregation and highly toxic, our study suggests that at least two mechanisms, namely to abolish RRM2 structure and to release CTD, may account for enhanced aggregation and toxicity of pathologically cleaved TDP-43.

Ameloblastin Peptides Modulates the Osteogenic Capacity of Human Mesenchymal Stem Cells.

During amelogenesis the extracellular enamel matrix protein AMBN is quickly processed into 17 kDa (N-terminus) and 23 kDa (C-terminus) fragments. In particular, alternatively spliced regions derived by exon 5/6 within the N-terminus region are known to be critical in biomineralization. Human mesenchymal stem cells (hMSC) also express and secrete AMBN, but it is unclear if this expression has effects on the hMSC themselves. If, as suggested from previous findings, AMBN act as a signaling molecule, such effects could influence hMSC growth and differentiation, as well as promoting the secretion of other signaling proteins like cytokines and chemokines. If AMBN is found to modulate stem cell behavior and fate, it will impact our understanding on how extracellular matrix molecules can have multiple roles during development ontogenesis, mineralization and healing of mesenchymal tissues. Here we show that synthetic peptides representing exon 5 promote hMSC proliferation. Interestingly, this effect is inhibited by the application of a 15 aa peptide representing the alternatively spliced start of exon 6. Both peptides also influence gene expression of RUNX2 and osteocalcin, and promote calcium deposition in cultures, indicating a positive influence on the osteogenic capacity of hMSC. We also show that the full-length AMBN-WT and N-terminus region enhance the secretion of RANTES, IP-10, and IL-8. In contrast, the AMBN C-terminus fragment and the exon 5 deleted AMBN (DelEx5) have no detectable effects on any of the parameters investigated. These findings suggest the signaling effect of AMBN is conveyed by processed products, whereas the effect on proliferation is differentially modulated through alternative splicing during gene expression.

ErbB4 cleavage by gonadotropin-releasing hormone receptor stimulation in cultured gonadotroph cells

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G-protein-coupled receptors, and its stimulation activates extracellular signal-regulated protein kinase (ERK). In the present study, we first examined the actions of GnRH on the ErbB family using two types of cultured gonadotroph cells. As reported previously, AG1478, an inhibitor of the ErbB family tyrosine kinase, inhibited GnRH-induced ERK activation in undifferentiated gonadotroph αT3-1 cells. However, AG1478 did not inhibit ERK activation in differentiated gonadotroph LβT2 cells, suggesting that transactivation of the ErbB family was not necessary for ERK activation in LβT2 cells. We found that ErbB4 was expressed in αT3-1 cells but not in LβT2 cells. GnRH induced the cleavage of ErbB4 and accumulation of an 80-kDa fragment in αT3-1 cells. Pharmacological experiments suggested that Gq/11 and tumor necrosis factor-α-converting enzyme (TACE) were essential for GnRH-induced ErbB4 cleavage. GnRH increased the phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS), indicating that GnRH activated protein kinase C (PKC). Down-regulation of PKC and bisindolylmaleimide I, a PKC inhibitor, strongly inhibited the GnRH-induced cleavage of ErbB4. It was surprising that GnRH treatment of LβT2 cells after overexpression of ErbB4 induced ErbB4 cleavage in a TACE-dependent manner. ErbB4 cleavage was induced also by treatment of αT3-1 cells, ErbB4-overexpressing LβT2 cells, and immortalized GnRH neurons (GT1-7 cells) with leuprorelin acetate. These results may suggest that the pharmacological effects of leuprorelin acetate are conducted through TACE-mediated proteolysis of membrane proteins, including ErbB4, in gonadotroph cells and GnRH neurons.

MET receptor variant R970C favors calpain-dependent generation of a fragment promoting epithelial cell scattering.

The receptor tyrosine kinase MET and its ligand, the hepatocyte growth factor, are essential to embryonic development, whereas deregulation of MET signaling is associated with tumorigenesis leading to various cancers, including lung carcinoma. Mutations in the MET kinase domain lead to constitutive kinase activity and are associated with tumorigenesis. In lung cancer, however, some mutations are found in the juxtamembrane domain, and their functional consequences are unknown. Because the juxtamembrane domain of MET is targeted by several proteolytic cleavages, involved in its degradation during cell death or under steady-state conditions, we evaluated the influence of these mutations on the MET proteolytic cleavages. In stably transfected epithelial cells expressing MET, the juxtamembrane mutations R970C, P991S, and T992I were found not to modify the known caspase or presenilin-dependent regulated intramembrane proteolysis. Yet when overexpressed, the R970C variant caused generation of an as yet undescribed 45-kDa fragment (p45 MET). This fragment was found in the confluent lung cancer cell line NCI-H1437 carrying the R970C mutation and at a lesser extent in cell lines expressing WT MET, suggesting that R970C mutation favors this cleavage. Generation of p45 MET required the activity of the calpain proteases, confirming the involvement of proteolysis. Ectopic expression of reconstituted p45 MET in epithelial cell lines favored cell scattering and invasion indicating active role of this fragment in HGF/SF induced responses. Hence, although the juxtamembrane mutations of MET do not affect its known proteolytic cleavages, the R970C MET variant favors calpain dependent proteolytic cleavage in lung cancer cells.

Expression of bovine rotavirus VP8 and preparation of IgY antibodies against recombinant VP8.

Group A rotavirus is the leading cause of acute gastroenteritis in cattle and swine. Although, vaccination against this virus is an effective strategy for prevention, additional strategy to control disease is necessary. Egg yolk immunoglobulin (IgY)-based passive immunization could be a better option in preventing this disease. Bovine rotavirus (BRV) is group A rotavirus and possesses a genome of 11 segments of double-stranded RNA. The outer layer of capsid is composed of two proteins (VP7 and VP4), which induce virus neutralizing antibodies. Trypsin cleavage of VP4 produces VP8 (28 kDa) and VP5 (60 kDa) fragments. Since a number of studies have demonstrated the induction of neutralizing antibodies using VP8 subunit vaccines, we have produced IgY against the recombinant VP8. The cDNA spanning the VP8 subunit was amplified from bovine rotavirus-infected cells and cloned into pET21d(+) expression vector to generate recombinant VP8. The resulting carboxy-terminal His-tagged VP8 proteins were expressed in Escherichia coli strain BL21(DE3) by isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. The recombinant proteins were purified using Ni-NTA agarose beads, and the purified protein was used as the immunizing agent to produce polyclonal antibodies in chicken. The resulting polyclonal antisera specifically recognized VP8 in Western blot assay and were able to neutralize BRV replication in cell cultures. These results demonstrate that IgY can be used in immunological assays and, in addition, in passive immunization of newborn calves against BRV.

Custom-Designed Affinity Capture LC-MS F(ab')2 Assay for Biotransformation Assessment of Site-Specific Antibody Drug Conjugates.

Affinity capture liquid chromatography-mass spectrometry (LC-MS) intact antibody assay has been widely used for direct drug-to-antibody ratio (DAR) and catabolite characterization of antibody-drug conjugates (ADCs). However, the intact mass spectra of new ADCs, which incorporate new types of linkers and payloads other than maytansines and auristatins, are more complex than those examined previously. The current method has showed some limitations in elucidating certain structural modifications. Herein, we report an alternative analytical approach for ADCs, such as THIOMAB antibody-drug conjugates (TDCs), where the linker drugs are site-specifically conjugated in the Fab region. The newly developed affinity capture LC-MS F(ab')2 assay incorporates affinity capture of human IgGs via binding to the Fab region, followed by on-bead IdeS digestion to remove the Fc domain specifically and uniformly. The resulting F(ab')2 (∼100 kDa) fragments contain the key ADC biotransformation information, such as drug-to-antibody ratio and drug metabolism and are more readily analyzed by electrospray ionization LC-MS than the intact ADC (∼150 kDa). The reduced size of analytes results in improved mass spectral sensitivity and resolution. In addition, the reduced and optimized sample preparation time, for example, rapid removal of the Fc fragment by IdeS digestion, minimizes assay artifacts of drug metabolism and skewed DAR profiles that may result from the prolonged incubation times (e.g., overnight enzymatic treatment for Fc deglycosylation). The affinity capture LC-MS F(ab')2 assay provides more detailed and accurate information on ADC biotransformations in vivo, enabling analysis of low-dose, labile, and complex site-specific ADCs with linker-drug conjugated in the Fab region.

Regulation of the blood-testis barrier by a local axis in the testis: role of laminin α2 in the basement membrane.

Laminin α2 is one of the constituent components of the basement membrane (BM) in adult rat testes. Earlier studies that used a mouse genetic model have shown that a deletion of laminin α2 impedes male fertility by disrupting ectoplasmic specialization (ES; a testis-specific, actin-rich anchoring junction) function along the length of Sertoli cell in the testis. This includes ES at the Sertoli cell-elongating/elongated spermatid interface, which is known as apical ES and possibly the Sertoli-Sertoli cell interface, known as basal ES, at the blood-testis barrier (BTB). Studies have also illustrated that there is a local regulatory axis that functionally links cellular events of spermiation that occur near the luminal edge of tubule lumen at the apical ES and the basal ES/BTB remodeling near the BM at opposite ends of the seminiferous epithelium during the epithelial cycle, known as the apical ES-BTB-BM axis. However, the precise role of BM in this axis remains unknown. Here, we show that laminin α2 in the BM serves as the crucial regulator in this axis as laminin α2, likely its 80-kDa fragment from the C terminus, was found to be transported across the seminiferous epithelium at stages VIII-IX of the epithelial cycle, from the BM to the luminal edge of the tubule, possibly being used to modulate apical ES restructuring at these stages. Of more importance, a knockdown of laminin α2 in Sertoli cells was shown to induce the Sertoli cell tight junction permeability barrier disruption via changes in localization of adhesion proteins at the tight junction and basal ES at the Sertoli cell BTB. These changes were found to be mediated by a disruption of F-actin organization that was induced by changes in the spatiotemporal expression of actin binding/regulatory proteins. Furthermore, laminin α2 knockdown also perturbed microtubule (MT) organization by considerable down-regulation of MT polymerization via changes in the spatiotemporal expression of EB1 (end-binding protein 1), a +TIP (MT plus-end tracking protein). In short, laminin α2 in the BM seems to play a crucial role in the BTB-BM axis by modulating BTB dynamics during spermatogenesis.-Gao, Y., Mruk, D., Chen, H., Lui, W.-Y., Lee, W. M., Cheng, C. Y. Regulation of the blood-testis barrier by a local axis in the testis: role of laminin α2 in the basement membrane.

Modifications of tau protein after cerebral ischemia and reperfusion in rats are similar to those occurring in Alzheimer’s disease – Hyperphosphorylation and cleavage of 4- and 3-repeat tau

Epidemiological studies have suggested a close relationship between cerebral ischemia and Alzheimer’s disease (AD). To clarify the pathological association of tau dynamics in both diseases, we performed comprehensive studies on the posttranslational modification of tau in cerebral ischemia and reperfusion (I/R) in rats. The present study suggests that both 4-repeat and 3-repeat tau isoforms are hyperphosphorylated in cerebral I/R, similar to the case in AD. The generation of a 60-kDa Asp421-truncated tau in cerebral I/R preceded the emergence of a 17-kDa 3-repeat tau fragment and a 25-kDa 4-repeat tau fragment. The regional redistribution of tau from the neuropil to neuronal perikarya in our stroke model is thought to share similarity with that occurring in AD. In addition, immunofluorescence staining revealed the formation of axonal varicosities in cerebral I/R. Altered tau distribution may influence microtubule stability, disturbances in axonal transport, and the resulting formation of axonal varicosities. The staining profiles of granules in the ischemic cortex that were immunopositive for RD3, RD4, and AT8 in neuronal perikarya and that were argyrophilic on Gallyas-Braak staining were similar to those in AD. These findings suggest that transient cerebral ischemia shares a common pathology with AD, in the modification of tau protein.

N-terminal fragment of cardiac myosin binding protein-C triggers pro-inflammatory responses in vitro

Myocardial infarction (MI) leads to loss and degradation of contractile cardiac tissue followed by sterile inflammation of the myocardium through activation and recruitment of innate and adaptive cells of the immune system. Recently, it was shown that cardiac myosin binding protein-C (cMyBP-C), a protein of the cardiac sarcomere, is degraded following MI, releasing a predominant N-terminal 40-kDa fragment (C0C1f) into myocardial tissue and the systemic circulation. We hypothesized that early release of C0C1f contributes to the initiation of inflammation and plays a key role in recruitment and activation of immune cells. Therefore, we investigated the role of C0C1f on macrophage/monocyte activation using both mouse bone marrow-derived macrophages and human monocytes. Here we demonstrate that C0C1f leads to macrophage/monocyte activation in vitro. Furthermore, C0C1f induces strong upregulation of pro-inflammatory cytokines (interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and interleukin-1β (IL-1β)) in cultured murine macrophages and human monocytes, resulting in a pro-inflammatory phenotype. We identified the toll-like receptor 4 (TLR4), toll-like receptor 2 (TLR2), and Advanced Glycosylation End Product-Specific Receptor (RAGE) as potential receptors for C0C1f whose activation leads to mobilization of the NFκB signaling pathway, a central mediator of the pro-inflammatory signaling cascade. Thus, C0C1f appears to be a key player in the initiation of inflammatory processes and might also play an important role upon MI.

CagA Phosphorylation in Helicobacter pylori-Infected B Cells Is Mediated by the Nonreceptor Tyrosine Kinases of the Src and Abl Families.

CagA is one of the most important virulence factors of the human pathogen Helicobacter pylori. CagA expression can be associated with the induction of severe gastric disorders such as gastritis, ulceration, gastric cancer, or mucosa-associated lymphoid tissue (MALT) lymphoma. After translocation through a type IV secretion system into epithelial cells, CagA is tyrosine phosphorylated by kinases of the Src and Abl families, leading to drastic cell elongation and motility. While the functional role of CagA in epithelial cells is well investigated, knowledge about CagA phosphorylation and its associated signal transduction pathways in B cells is only marginal. Here, we established the B cell line MEC1 derived from a B cell chronic lymphocytic leukemia (B-CLL) patient as a new infection model to study the signal transduction in B cells controlled by H. pylori. We observed that CagA was rapidly injected, strongly tyrosine phosphorylated, and cleaved into a 100-kDa N-terminal and a 40-kDa C-terminal fragment. To identify upstream signal transduction pathways of CagA phosphorylation in MEC1 cells, pharmacological inhibitors were employed to specifically target Src and Abl kinases. We observed that CagA phosphorylation was strongly inhibited upon treatment with an Src inhibitor and slightly diminished when the Abl kinase inhibitor imatinib mesylate (Gleevec) was applied. The addition of dasatinib to block c-Abl and Src kinases led to a complete loss of CagA phosphorylation. In conclusion, these results demonstrate an important role for Src and Abl tyrosine kinases in CagA phosphorylation in B cells, which represent druggable targets in H. pylori-mediated gastric MALT lymphoma.

Osteopontin Fragments with Intact Thrombin-Sensitive Site Circulate in Cervical Cancer Patients.

We investigated whether circulating osteopontin (OPN) could be used as a biomarker for cervical cancer. We employed a monoclonal antibody (mAb 659) specific for the unique and intact thrombin-sensitive site in OPN using an inhibition ELISA. We found significantly higher levels of OPN in 33 cervical cancer patients in both the plasma (mean +/- SD, 612 +/- 106 ng/mL) and serum (424 +/- 121 ng/mL) compared to healthy subjects [409 +/- 56 ng/mL, from 31 plasma samples (P < 0.0001), and 314 +/- 98 ng/mL, from 32 serum samples (P = 0.0002), respectively]. Similar results were obtained when the plasma from a bigger group (147 individuals) of cervical cancer patients (560 +/- 211 ng/mL) were compared with the same plasma samples of the healthy individuals (P = 0.0014). More significantly, the OPN level was highest in stage III-IV disease (614 +/- 210 ng/mL, from 52 individuals; P = 0.0001) and least and non-discriminatory in stage I (473 +/- 110 ng/mL, from 40 individuals; P = 0.5318). No such discrimination was found when a mAb of a different specificity (mAb 446) was used in a similar inhibition ELISA to compare the two groups in the first study; a commercial capture ELISA also failed. The possibility that the target epitope recognized by the antibody probe in these assays was absent from the circulating OPN due to protein truncation was supported by gel fractionation of the OPN found in patients’ plasma: 60–64 kDa fragments were found instead of the presumably full-length OPN (68 kDa) seen in healthy people. How these fragments are generated and what possible role they play in cancer biology remain interesting questions.

Abstract 95: The Role of TGFβ Signaling in a Fibrotic cMyBP-C HCM/HF Model

Purpose: Hypertrophic cardiomyopathy (HCM) is considered one of the most common genetic heart disorders with a prevalence of about 1 in 500 people, with 35% of those affected being attributed to mutations within the gene encoding cardiac myosin-binding protein C (cMyBP-C). Cardiac stress, as well as cMyBP-C mutations, can trigger production of a 40kDa truncated fragment derived from the amino terminus of cMyBP-C. Genetic expression of this 40kDa fragment in mouse cardiomyocytes ( Mybp3 40kDa ) leads to HCM, fibrosis and heart failure, mimicking human disease progression. The transforming growth factor-β (TGFβ) signaling pathway has been implicated in a variety of fibrotic processes. The goal of this study is to define the role of TGFβ signaling in distinct cell populations, the cardiomyocyte and fibroblast, in the cMyBP-C HCM/HF model. Methods and results: Masson’s Trichrome staining, PCR arrays, immunohistochemistry and western blots were performed to characterize the fibrotic progression in Mybp3 40kDa transgenic mice. Cardiac fibrosis was initially detected 4 weeks after transgene expression. Extensive interstitial fibrosis and severe atrial fibrosis were detected at 16 weeks. Both canonical and non-canonical TGFβ pathways were active during fibrotic progression. To specifically block TGFβ signaling in Mybp3 40kDa transgenic mice, compound mutant mice were generated, in which the tgfbr1 or tgfbr2 alleles were ablated, either in cardiomyocytes or in activated fibroblasts (myofibroblasts) by αMHC-Cre or Periostin-MerCreMer-Cre respectively. Blockage of TGFβ signaling in either cardiomyocytes or myofibroblasts alleviated cardiac fibrosis. Furthermore, treatment with the non-canonical TGFβ signaling inhibitor MMI-0100 also alleviated cardiac fibrosis and increased the life span of the Mybp3 40kDa transgenic mice. Conclusions: TGFβ signaling is activated in the Mybp3 40kD HCM/HF model. Genetic or pharmaceutical inhibition of TGFβ signaling inhibited fibrosis and increased the life span in this model.

Healthy and Osteoarthritic Synovial Fibroblasts Produce a Disintegrin and Metalloproteinase with Thrombospondin Motifs 4, 5, 7, and 12: Induction by IL-1β and Fibronectin and Contribution to Cartilage Damage

Current description of osteoarthritis includes the involvement of synovial inflammation. Studies contributing to understanding the mechanisms of cross-talk and feedback among the joint tissues could be relevant to the development of therapies that block disease progression. During osteoarthritis, synovial fibroblasts exposed to anomalous mechanical forces and an inflammatory microenvironment release factors such as a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) metalloproteinases that mediate tissue damage and perpetuate inflammation. We therefore studied the production of ADAMTS by synovial fibroblasts and their contribution to cartilage degradation. Moreover, we analyzed the implication of two mediators present in the osteoarthritis joint, IL-1β as proinflammatory cytokine, and 45-kDa fibronectin fragments as products of matrix degradation. We reported that synovial fibroblasts constitutively express and release ADAMTS 4, 5, 7, and 12. Despite the contribution of both mediators to the stimulation of Runx2 and Wnt/β-catenin signaling pathways, as well as to ADAMTS expression, promoting the degradation of aggrecan and cartilage oligomeric matrix protein from cartilage, fibronectin fragments rather than IL-1β played the major pathological role in osteoarthritis, contributing to the maintenance of the disease. Moreover, higher levels of ADAMTS 4 and 7 and a specific regulation of ADAMTS-12 were observed in osteoarthritis, suggesting them as new potential therapeutic targets. Therefore, synovial fibroblasts provide the biochemical tools to the chronicity and destruction of the osteoarthritic joints.

Accumulation of human full-length tau induces degradation of nicotinic acetylcholine receptor α4 via activating calpain-2.

Cholinergic impairments and tau accumulation are hallmark pathologies in sporadic Alzheimer’s disease (AD), however, the intrinsic link between tau accumulation and cholinergic deficits is missing. Here, we found that overexpression of human wild-type full-length tau (termed hTau) induced a significant reduction of α4 subunit of nicotinic acetylcholine receptors (nAChRs) with an increased cleavage of the receptor producing a ~55kDa fragment in primary hippocampal neurons and in the rat brains, meanwhile, the α4 nAChR currents decreased. Further studies demonstrated that calpains, including calpain-1 and calpain-2, were remarkably activated with no change of caspase-3, while simultaneous suppression of calpain-2 by selective calpain-2 inhibitor but not calpain-1 attenuated the hTau-induced degradation of α4 nAChR. Finally, we demonstrated that hTau accumulation increased the basal intracellular calcium level in primary hippocampal neurons. We conclude that the hTau accumulation inhibits nAChRs α4 by activating calpain-2. To our best knowledge, this is the first evidence showing that the intracellular accumulation of tau causes cholinergic impairments.

Calpain-Mediated Cleavage of Calcineurin in Puromycin Aminonucleoside-Induced Podocyte Injury.

The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus are widely used in the treatment of proteinuria diseases. As the direct target of these drugs, calcineurin has previously been demonstrated to play a role in proteinuria diseases. However, aside from its immune-related effects, the local status of calcineurin in renal inherent cells has not been fully explored in the settings of proteinuria disease and podocyte injury. In this study, calcineurin activity and protein expression in the well-known puromycin aminonucleoside (PAN)-induced podocyte injury model were examined. Interestingly, we found that calcineurin activity was abnormally increased in PAN-treated podocytes, whereas the expression of the full-length 60-kDa calcineurin protein was decreased. This result suggests that there may be another activated form of calcineurin that is independent of the full-length phosphatase. To investigate whether calpain is involved in regulating calcineurin, we exposed PAN-treated podocytes to both pharmacological inhibitors of calpain and specific siRNAs against calpain. Calpain blockade reduced the enhanced calcineurin activity and restored the down-regulated expression of 60-kDa calcineurin. In addition, purified calpain protein was incubated with podocyte extracts, and a 45-kDa fragment of calcineurin was identified; this finding was confirmed in PAN-induced podocyte injury and calpain inhibition experiments. We conclude that calcineurin activity is abnormally increased during PAN-induced podocyte injury, whereas the expression of the full-length 60-kDa calcineurin protein is down-regulated due to over-activated calpain that cleaves calcineurin to form a 45-kDa fragment.