Durba Mukhopadhyay

Impaired cumulus mucification and female sterility in tumor necrosis factor-induced protein-6 deficient mice

Mucification of the cumulus layer around the oocyte is an obligatory process for female fertility. Tumor necrosis factor-induced protein-6 (TNFIP6 or TSG6) has been shown to be specifically expressed during this process. We have generated TNFIP6-deficient mice and tested the ability of their cumulus cells to undergo mucification. Cumulus cell-oocyte complexes fail to expand in TNFIP6-deficient female mice because of the inability of the cumulus cells to assemble their hyaluronan-rich extracellular matrix. The impaired cumulus matrix formation is due to the lack of covalent complexes between hyaluronan and the heavy chains of the inter-α-trypsin inhibitor family. As a consequence, TNFIP6-deficient females are sterile. Cultured TNFIP6-deficient cumulus cell-oocyte complexes also fail to expand when stimulated with dibutyryl cyclic AMP or epidermal growth factor. Recombinant TNFIP6 is able to catalyze the covalent transfer of heavy chains to hyaluronan in a cell-free system, restore the expansion of Tnfip6-null cumulus cell-oocyte complexes in vitro, and rescue the fertility in Tnfip6-null females. These results provide clear evidence that TNFIP6 is a key catalyst in the formation of the cumulus extracellular matrix and indispensable for female fertility.

Primary Murine Airway Smooth Muscle Cells Exposed to Poly(I,C) or Tunicamycin Synthesize a Leukocyte-adhesive Hyaluronan Matrix

Asthmatic attacks often follow viral infections with subsequent airway smooth muscle cell proliferation and the formation of an abnormal hyaluronan extracellular matrix with infiltrated leukocytes. In this study, we show that murine airway smooth muscle cells (MASM) treated with polyinosinic acid-polycytidylic acid (poly(I,C)), a double-stranded RNA that simulates a viral infection, synthesize an abnormal hyaluronan matrix that binds leukocytes (U937 cells). Synthesis of this matrix is initiated rapidly and accumulates linearly for ∼10 h, reaching a plateau level ∼7-fold higher than control cultures. MASM cells treated with tunicamycin, to induce endoplasmic reticulum stress, also rapidly initiate synthesis of the abnormal hyaluronan matrix with linear accumulation for ∼10 h, but only reach a plateau level ∼2-fold higher than control cultures. In contrast to poly(I,C), the response to tunicamycin depends on cell density, with pre-confluent cells producing more abnormal matrix per cell. Furthermore, U937 cell adhesion per hyaluronan content is higher in the sparse matrix produced in response to tunicamycin, suggesting that the structure in the poly(I,C)-induced matrix masks potential binding sites. When MASM cells were exposed to tunicamycin and poly(I,C) at the same time, U937 cell adhesion was partially additive, implying that these two toxins stimulate hyaluronan synthesis through two different pathways. We also characterized the size of hyaluronan produced by MASM cells, in response to poly(I,C) and tunicamycin, and we found that it ranges from 1500 to 4000 kDa, the majority of which was ∼4000 kDa and not different in size than hyaluronan made by untreated cells.

Differentiated Murine Airway Epithelial Cells Synthesize a Leukocyte-adhesive Hyaluronan Matrix in Response to Endoplasmic Reticulum Stress

In this report, we describe a novel method for culturing murine trachea epithelial cells on a native basement membrane at an air-liquid interface to produce a pseudostratified, differentiated airway epithelium composed of ciliated and nonciliated cells. This model was used to examine hyaluronan synthesis by the airway epithelial cells (AECs) in response to poly(I,C) and tunicamycin. The former induces a response similar to viral infection, and the latter is a bacterial toxin known to induce endoplasmic reticulum (ER) stress. We found significant accumulation of hyaluronan on the apical surface of the AECs in response to ER stress, but, unlike previously reported results with smooth muscle cells, no increase in hyaluronan was observed in response to poly(I,C). Monocytic U937 cells adhered at 4 °C to the apical surface of the AECs subjected to ER stress by a mechanism almost entirely mediated by hyaluronan. The U937 cells spontaneously released themselves from the abnormal hyaluronan matrix when their metabolism was restored by shifting the temperature from 4 to 37 °C in a custom-made flow chamber. Time lapse confocal microscopy permitted live imaging of this interaction between the U937 cells and the hyaluronan matrix and their subsequent response at 37 °C. Within 45 min, we observed dynamic protrusions of the U937 cell plasma membrane into nearby hyaluronan matrix, resulting in the degradation of this matrix. Simultaneously, we observed some reorganization of the hyaluronan matrix, from a generalized, apical distribution to localized regions around the AEC tight junctions. We discuss the implications these results might have for the airway epithelium and its relation to airway inflammation and hyperresponsiveness associated with asthma and other airway diseases.

Airway Smooth Muscle Cells Synthesize Hyaluronan Cable Structures Independent of Inter-α-inhibitor Heavy Chain Attachment

The covalent association of inter-α-inhibitor-derived heavy chains (HCs) with hyaluronan was first described in synovial fluid from arthritic patients and later described as a structural and functional component of hyaluronan “cable” structures produced by many different cells and stimuli. HC transfer has been shown to be mediated by the protein product of TSG-6 (tumor necrosis factor-stimulated gene 6). Considering the accumulation of hyaluronan in airways following asthmatic attacks and the subsequent infiltration of leukocytes, we sought to characterize HC substitution of hyaluronan “cables” in primary mouse airway smooth muscle cells (MASM) and primary human airway smooth muscle cells (HASM). We found that cells derived from mice lacking TSG-6 had no defect in hyaluronan production or hyaluronan-mediated leukocyte adhesion when treated with the viral mimic poly(I,C). Functional hyaluronan cables were induced by cycloheximide in the confirmed absence of protein synthesis, with or without simultaneous treatment with poly(I,C). We characterized the species specificity of the antibody other investigators used to describe the HC-hyaluronan complex of hyaluronan cables and found minimal affinity to bovine-derived HCs in contrast to HCs from mouse and human sera. Thus, we cultured MASM and HASM cells in serum from these three sources and analyzed hyaluronan extracts for HCs and other hyaluronan-binding proteins, using parallel cumulus cell-oocyte complex (COC) extracts as positive controls. We conclude that, if hyaluronan cables derived from MASM and HASM cells are substituted with HCs, the amount of substitution is significantly below the limit of detection when compared with COC extracts of similar hyaluronan mass.

Regulation of heparan sulfate and chondroitin sulfate glycosaminoglycan biosynthesis by 4-fluoro-glucosamine in murine airway smooth muscle cells

The importance of the pathological changes in proteoglycans has driven the need to study and design novel chemical tools to control proteoglycan synthesis. Accordingly, we tested the fluorinated analogue of glucosamine (4-fluoro-N-acetyl-glucosamine (4-F-GlcNAc)) on the synthesis of heparan sulfate (HS) and chondroitin sulfate (CS) by murine airway smooth muscle (ASM) cells in the presence of radiolabeled metabolic precursors. Secreted and cell-associated CS and HS were assessed for changes in size by Superose 6 chromatography. Treatment of ASM cells with 4-F-GlcNAc (100 μm) reduced the quantity (by 64.1–76.6%) and decreased the size of HS/CS glycosaminoglycans associated with the cell layer (Kav shifted from 0.30 to 0.45). The quantity of CS secreted into the medium decreased by 65.7–73.0%, and the size showed a Kav shift from 0.30 to 0.50. Treatment of ASM cells with 45 μm and 179 μm 4-F-GlcNAc in the presence of a stimulator of CS synthesis, 4-methylumbelliferyl-β-d-xyloside, reduced the amount of the xyloside-CS chains by 65.4 and 87.0%, respectively. The size of xyloside-CS chains synthesized in the presence of 4-F-GlcNAc were only slightly larger than those with xyloside treatment alone (Kav of 0.55 compared with that of 0.6). The effects of 4-F-GlcNAc to inhibit CS synthesis were not observed with equimolar concentrations of glucosamine. We propose that 4-F-GlcNAc inhibits CS synthesis by inhibiting 4-epimerization of UDP-GlcNAc to UDP-GalNAc, thereby depleting one of the substrates required, whereas HS elongation is inhibited by truncation when the nonreducing terminus of the growing chain is capped with 4-F-GlcNAc.