Dina Montufar-Solis

Calreticulin, PDI, Grp94 and BiP chaperone proteins are associated with retained COMP in pseudoachondroplasia chondrocytes

Cartilage oligomeric matrix protein (COMP), a large pentameric glycoprotein and member of the thrombospondin (TSP) group of extracellular proteins, is found in the territorial matrix surrounding chondrocytes. More than 50 unique COMP mutations have been identified as causing two skeletal dysplasias: pseudoachondroplasia (PSACH); and multiple epiphyseal dysplasia (EDM1). Recent studies suggest that calcium-binding and calcium-induced protein folding differ between wild type and mutant proteins, and abnormal processing of the mutant COMP protein contributes to the characteristic enlarged lamellar appearing rER cisternae in PSACH and EDMI chondrocytes in vivo and in vitro. Towards the goal of delineating the pathogenesis of PSACH and EDM1, in-vivo PSACH growth plate and in-vitro PSACH chondrocytes cultured in alginate beads were examined to identify and localize the chaperone proteins participating in the processing of the retained extracellular matrix proteins in the PSACH rER. Aggrecan was localized to both the rER cisternae and matrix while COMP and type IX collagen were only found in the rER. Type II collagen was solely found in the ECM suggesting that it is processed and transported differently from other retained ECM proteins. Five chaperone proteins: BiP (Grp78); calreticulin (CRT); protein disulfide (PDI); ERp72; and Grp94, demonstrated immunoreactivity in the enlarged PSACH cisternae and the short rER channels of chondrocytes from both in-vivo and in-vitro samples. The chaperone proteins cluster around the electron dense material within the enlarged rER cisternae. CRT, PDI and GRP94 AB-gold particles appear to be closely associated with COMP. Immunoprecipitation and Western blot, and Fluorescence Resonance Energy Transfer (FRET) analyses indicate that CRT, PDI and GRP94 are in close proximity to normal and mutant COMP and BiP to mutant COMP. These results suggest that these proteins play a role in the processing and transport of wild type COMP in normal chondrocytes and in the retention of mutant COMP in PSACH chondrocytes.

Retention of cartilage oligomeric matrix protein (COMP) and cell death in redifferentiated pseudoachondroplasia chondrocytes.

Cartilage oligomeric matrix protein (COMP) is a large extracellular glycoprotein that is found in the territorial matrix surrounding chondrocytes. Two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) are caused by mutations in the calcium binding domains of COMP. In this study, we identified two PSACH mutations and assessed the effect of these mutations on redifferentiated chondrocyte structure and function. We confirmed, in vitro, that COMP is retained in enormous cisternae of the rough endoplasmic reticulum (rER) and relatively absent in the PSACH matrix. The rER accumulation may compromise chondrocyte function, leading to chondrocyte death. Moreover, while COMP appears to be deficient in the PSACH matrix, the matrix appeared to be normal but the over-all quantity was reduced. These results suggest that the abnormality in linear growth in PSACH may result from decreased chondrocyte numbers which would also affect the amount of matrix produced.

Selective Upregulation of microRNA Expression in Peripheral Blood Leukocytes in IL-10 −/− Mice Precedes Expression in the Colon

IL-10−/− mice, an animal model of Th1-mediated inflammatory bowel disease, were screened for the expression of 600 microRNAs (miRNAs) using colonic tissues and PBLs from animals having either mild inflammation or severe intestinal inflammation. The development of colonic inflammation in IL-10−/− mice was accompanied by upregulation in the expression of 10 miRNAs (miR-19a, miR-21, miR-31, miR-101, miR-223, miR-326, miR-142-3p, miR-142-5p, miR-146a, and miR-155). Notably, the expression of all of these miRNAs plus miR-375 was elevated in PBLs of IL-10−/− mice at a time when colonic inflammation was minimal, suggesting that changes in specific miRNAs in circulating leukocytes may be harbingers of ensuing colonic pathology. In vitro exposure of colonic intraepithelial lymphocytes to IL-10 resulted in downregulation of miR-19a, miR-21, miR-31, miR-101, miR-223, and miR-155. Interestingly, unlike IL-10−/− mice, changes in miRNAs in PBL of dextran sulfate sodium-treated mice were minimal but selectively elevated in the colon after pathology was severe. We further show that miR-223 is a negative regulator of the Roquin ubiquitin ligase, Roquin curtails IL-17A synthesis, and the 3′ untranslated region of Roquin is a target for miR-223, thus defining a molecular pathway by which IL-10 modulates IL-17–mediated inflammation. To identify additional miRNAs that may be involved in the regulation of Roquin, transcriptome analysis was done using cDNAs from HeLa cells transfected with 90 miRNA mimics. Twenty-six miRNAs were identified as potential negative regulators of Roquin, thus demonstrating functional complexity in gene expression regulation by miRNAs.

T‐cell activation in the intestinal mucosa

Summary:  The vast majority of peripheral T cells exist as resting lymphocytes until a signal for activation has been received. In response to antigen, this activation involves ligation of the T‐cell receptor (TCR) and signal transmission through the CD3 complex, which then initiates a cascade of intracellular events that lead to the expression of genes used in T‐cell activation. T‐cell activation also requires soluble mediators in the form of cytokines and chemokines that regulate the process in both positive and negative ways, and costimulatory signals received in conjunction with TCR/CD3 signaling are important in the activation of T cells. Unlike T cells in other peripheral immune compartments, small and large intestinal intraepithelial lymphocytes (IELs) bear some but not all properties of activated T cells, suggesting that they constitute a large population of ‘partially activated’ effector cells. Thus, regulation of the IEL activation process must be held in tight check, yet it must be ready to respond to foreign antigen rapidly and effectively. We discuss how costimulatory molecules may hold the key to controlling IEL activation through a multiphase process beginning with cells that have already entered into the early stage of activation.

Bone marrow cells produce a novel TSHβ splice variant that is upregulated in the thyroid following systemic virus infection

Although cells of the immune system can produce thyroid-stimulating hormone (TSH), the significance of that remains unclear. Using 5′ rapid amplification of cDNA ends (RACE), we show that mouse bone marrow (BM) cells produce a novel in-frame TSHβ splice variant generated from a portion of intron 4 with all of the coding region of exon 5, but none of exon 4. The TSHβ splice variant gene was expressed at low levels in the pituitary, but at high levels in the BM and the thyroid, and the protein was secreted from transfected Chinese hamster ovary (CHO) cells. Immunoprecipitation identified an 8 kDa product in lysates of CHO cells transfected with the novel TSHβ construct, and a 17 kDa product in lysates of CHO cells transfected with the native TSHβ construct. The splice variant TSHβ protein elicited a cAMP response from FRTL-5 thyroid follicular cells and a mouse alveolar macrophage (AM) cell line. Expression of the TSHβ splice variant, but not the native form of TSHβ, was significantly upregulated in the thyroid during systemic virus infection. These studies characterize the first functional splice variant of TSHβ, which may contribute to the metabolic regulation during immunological stress, and may offer a new perspective for understanding autoimmune thyroiditis.

Cytoskeletal abnormalities in chondrocytes with EXT1 and EXT2 mutations.

The EXT genes are a group of putative tumor suppressor genes that previously have been shown to participate in the development of hereditary multiple exostoses (HME), HME‐associated and isolated chondrosarcomas. Two HME disease genes, EXT1 and EXT2, have been identified and are expressed ubiquitously. However, the only known effect of mutations in the EXT genes is on chondrocyte function as evidenced by aberrant proliferation of chondrocytes leading to formation of bony, cartilage‐capped projections (exostoses). In this study, we have characterized exostosis chondrocytes from three patients with HME (one with EXT1 and two with EXT2 germline mutations) and from one individual with a non‐HME, isolated exostosis. At the light microscopic level, exostosis chondrocytes have a stellate appearance with elongated inclusions in the cytoplasm. Confocal and immunofluorescence of in vitro and in vivo chondrocytes showed that these massive accumulations are composed of actin bundled by 1.5‐μm repeat cross‐bridges of α‐actinin. Western blot analysis shows that exostosis chondrocytes from two out of three patients aberrantly produce high levels of muscle‐specific α‐actin, whereas β‐actin levels are similar to normal chondrocytes. These findings suggest that mutations in the EXT genes cause abnormal processing of cytoskeleton proteins in chondrocytes.

ICOS promotes IL‐17 synthesis in colonic intraepithelial lymphocytes in IL‐10−/− mice

In the absence of IL‐10, colonic inflammation ensues, which is characterized by high levels of IL‐17. Here, we demonstrate a direct correlation between ICOS expression and IL‐17 production in cIELs. IL‐10−/− mice had increased numbers of cIELs and greater colon weight. Although the CD69 early activation antigen was expressed on cIELs from normal and IL‐10−/− mice, ICOS was expressed only on cIELs from IL‐10−/− mice. IL‐17‐producing cells in IL‐10−/− mice consisted of CD4+ and CD8+ cIELs; however, CD4+ cells were the predominant IL‐17‐producing cell population. Culture of cIELs from IL‐10−/− mice with IL‐23 resulted in an increase in ICOS and IL‐17 expression, whereas IL‐10 suppressed expression of ICOS and IL‐17. This occurred in primary cultures and recall stimulation experiments. The ICOS ligand B7RP‐1 was up‐regulated on colonic epithelial cells and on a population of large granular leukocytes during inflammation. Culture of cIELs with B7RP‐1+ DCs enhanced IL‐17A production from normal cIELs but failed to do so using cIELs from ICOS−/− mice. In vivo treatment of IL‐10−/− mice with antibody to ICOS resulted in a significant reduction in colonic pathology. These findings implicate ICOS as an activational signal of Th17 cells during chronic intestinal inflammation, and they suggest that under some conditions, control of ICOS expression may help to suppress chronic intestinal inflammation.

Using cartilage to repair bone: an alternative approach in tissue engineering.

Materials and techniques currently used for bone replacement/repair conform to the current paradigm, relying on bone or bone products to produce bone or induce bone formation. Yet, nature forms and heals most of the skeleton by ossification of a cartilaginous model. In this study, we cultured aggregates of E10.5 or E12 mouse embryonic limb cells in the bioreactor for 3 weeks, determined the stages of cartilage differentiation attained, and assessed the ossification and bone healing potential of the spheroids by implantation adjacent to, or directly in, a skull defect. Cultured spheroids had large cartilaginous areas, sometimes with cellular arrangements characteristic of growth plate zones. Aggregates implanted for 2 weeks adjacent to a defect mineralized and ossified (histology, micro-CT). Defects with implants had a central mass of differentiated and differentiating bone, with osteoclast activity, filling the defect. Controls had considerable remodeling on the bone edges demarcating the still present defect. This study shows that cartilage, grown in the bioreactor for 3 weeks, ossified when implanted adjacent to a bone defect, and when implanted directly in a defect, contributed to its healing. Our ability to grow differentiated bone-forming cartilage for implantation is an alternative approach in the field of bone repair.

Differential Cytokine Patterns in Mouse Macrophages and Gingival Fibroblasts After Stimulation With Porphyromonas gingivalis or Escherichia coli Lipopolysaccharide

BACKGROUND A major cause of chronic inflammatory periodontal disease is Porphyromonas gingivalis, a non-motile, Gram-negative, rod-shaped, anaerobic bacterium. Within gingival tissue, both macrophages and fibroblasts participate in the immune response to foreign entities by releasing cytokines and expressing molecules to recruit and activate lymphocytes. However, the contribution of gingival macrophages and fibroblasts to the immune response to P. gingivalis infection is not fully known. METHODS The AMJ2-C8 cell line (AM cells), a mouse alveolar macrophage cell line, and ESK-1 cells, a mouse gingival fibroblast cell line made in our laboratory, were treated with lipopolysaccharide (LPS) from either P. gingivalis or Escherichia coli. The expression of immune response molecules was quantified by real-time polymerase chain reaction and enzyme-linked immunoassay. RESULTS AM and ESK-1 cells responded differently to P. gingivalis and E. coli LPS stimulation. The ESK-1 gingival fibroblast cell line was more responsive to E. coli LPS stimulation as seen by elevated levels of interleukin (IL)-6, inducible nitric oxide, and monocyte chemotactic protein-1 expression relative to stimulation by P. gingivalis LPS. Conversely, the AM macrophage cell line was more responsive to P. gingivalis LPS stimulation, particularly for interleukin IL-1β, IL-6, and monocyte chemotactic protein-1, relative to stimulation by E. coli LPS. CONCLUSION These findings demonstrate that E. coli LPS induces a stronger cytokine and chemokine response in gingival fibroblasts, whereas P. gingivalis LPS induces a stronger response in macrophages.

Small intestine inflammation in Roquin-mutant and Roquin-deficient mice.

Roquin, an E3 ubiquitin ligase that localizes to cytosolic RNA granules, is involved in regulating mRNA stability and translation. Mice that have a M199R mutation in the Roquin protein (referred to as sanroque or Roquinsan/san mice) develop autoimmune pathologies, although the extent to which these occur in the intestinal mucosa has not been determined. Here, we demonstrate that Roquinsan/san mice reproducibly develop intestinal inflammation in the small intestine but not the colon. Similarly, mice generated in our laboratory in which the Roquin gene was disrupted by insertion of a gene trap cassette (Roquingt/gt mice) had small intestinal inflammation that mimicked that of Roquinsan/san mice. MLN cells in Roquinsan/san mice consisted of activated proliferating T cells, and had increased numbers of CD44hi CD62Llo KLRG1+ short-lived effector cells. Proportionally more small intestinal intraepithelial lymphocytes in Roquinsan/san mice expressed the ICOS T cell activation marker. Of particular interest, small intestinal lamina propria lymphocytes in Roquinsan/san mice consisted of a high proportion of Gr-1+ T cells that included IL-17A+ cells and CD8+ IFN-γ+ cells. Extensive cytokine dysregulation resulting in both over-expression and under-expression of chemotactic cytokines occurred in the ileum of Roquinsan/san mice, the region most prone to the development of inflammation. These findings demonstrate that chronic inflammation ensues in the intestine following Roquin alteration either as a consequence of protein mutation or gene disruption, and they have implications for understanding how small intestinal inflammation is perpetuated in Crohns disease (CD). Due to the paucity of animal models of CD-like pathophysiology in the small intestine, and because the primary gene/protein defects of the Roquin animal systems used here are well-defined, it will be possible to further elucidate the underlying genetic and molecular mechanisms that drive the disease process.