Identification of Tctex2beta, a novel dynein light chain family member that interacts with different transforming growth factor-beta receptors.

Endoglin is a membrane-inserted protein that is preferentially synthesized in angiogenic vascular endothelial and smooth muscle cells. Endoglin associates with members of the transforming growth factor-beta (TGF-beta) receptor family and has been identified as the gene involved in hereditary hemorrhagic telangiectasia. Although endoglin is known to affect cell responses to TGF-beta, its mode of action is largely unknown. We performed yeast two-hybrid screening of a human placental cDNA library and isolated a new endoglin-binding partner, a novel 221-amino acid member of the Tctex1/2 family of cytoplasmic dynein light chains named Tctex2beta, as the founder of a new Tctex1/2 subfamily. The interaction was localized exclusively to the cytoplasmic domain of endoglin. Reverse transcription-PCR showed expression of Tctex2beta in a wide range of tissues, including vascular endothelial and smooth muscle cells, placenta, and testis, as well as in several tumor cell lines. High expression levels were found in human umbilical vein endothelial cells and the large cell lung cancer cell line. Forced expression of Tctex2beta had a profound inhibitory effect on TGF-beta signaling. Additional Tctex2beta-interacting receptors were identified to be the TGF-beta type II receptor and most likely beta-glycan, but not ALK5, ALK1, or the bone morphogenetic protein type II receptor. Upon fluorescence tagging, co-localization of Tctex2beta and endoglin, as well as Tctex2beta, endoglin, and the TGF-beta type II receptor, was observed by different microscopy techniques. Our findings link endoglin for the first time to microtubule-based minus end-directed transport machinery, suggesting that some endoglin functions might be regulated and directed by its interaction with the cytoplasmic dynein light chain Tctex2beta.

Generation of a vector system facilitating cloning of DMBT1 variants and recombinant expression of functional full-length DMBT1.

Deleted in malignant brain tumours 1 (DMBT1) codes for a approximately 340kDa glycoprotein with highly repetitive scavenger receptor cysteine-rich (SRCR) domains. DMBT1 was implicated in cancer, defence against viral and bacterial infections, and differentiation of epithelial cells. Recombinant expression and purification of DMBT1 is an essential step for systematic standardized functional research and towards the evaluation of its therapeutical potential. So far, DMBT1 is obtained from natural sources such as bronchioalveolar lavage or saliva, resulting in time consuming sample collection, low yields, and protein preparations which may substantially vary due to differential processing and genetic polymorphism, all of which impedes functional research on DMBT1. Cloning of DMBT1 cDNAs is hampered because of the size and the 13 highly homologous SRCR exons. In this study, we report on the setup of a vector system that facilitates cloning of DMBT1 variants. We demonstrate applicability of the vector system by expression of the largest DMBT1 variant in a tetracycline-inducible mammalian expression system using the Chinese hamster ovary cell line. Yields up to 30 mg rDMBT1 per litre of cell culture supernatant could be achieved with an optimized production procedure. By harnessing the specific bacteria-binding property of DMBT1 we established an affinity purification procedure which allows the isolation of more than 3 mg rDMBT1 with a purity of about 95%. Although the glycosylation moieties of rDMBT1 are different from DMBT1(SAG) isolated from saliva, we demonstrate that rDMBT1 is functionally active in aggregating Gram-positive and Gram-negative bacteria and binding to C1q and lactoferrin, which represent two known endogenous DMBT1 ligands.

Subcellular localization and in vivo subunit interactions of ubiquitous mu-calpain.

Ubiquitously expressed calpains are Ca(2+)-dependent, intracellular cysteine proteases comprising a large catalytic subunit (domains DI-DIV) and a noncovalently bound small regulatory subunit (domains DV and DVI). It is unclear whether Ca(2+)-induced calpain activation is followed by subunit dissociation or not. Here, we have applied advanced fluorescence microscopy techniques to study calpain subunit interactions in living cells using recombinant calpain subunits or domains fused to enhanced cyan and enhanced yellow fluorescent reporter proteins. All of the overexpressed variants of the catalytic subunit (DI-IV, DI-III, and DI-IIb) were active and Ca(2+)-dependent. The intact large subunit, but not its truncated variants, associates with the small subunit under resting and ionomycin-activated conditions. All of the variants were localized in cytoplasm and nuclei, except DI-IIb, which accumulates in the nucleus and in nucleoli as shown by microscopy and cell fractionation. Localization studies with mutated and chimeric variants indicate that nuclear targeting of the DI-IIb variant is conferred by the two N-terminal helices of DI. Only those variants that contain DIII migrated to membranes upon the addition of ionomycin, suggesting that DIII is essential for membrane targeting. We propose that intracellular localization and in particular membrane targeting of activated calpain, but not dissociation of its intact subunits, contribute to regulate its proteolytic activity in vivo.

The role of contractile microfilaments in the morphogenesis of the developing foregut of chick embryos.

The cellular processes that lead to changes in shape (morphogenesis) and organ formation (organogenesis) are poorly understood. Local contraction of microfilaments can change cell shape and lead to changes of tissue shape. To clarify the role of contractile microfilaments in the foregut morphogenesis of chick embryos, 2- to 4-day-old embryos were exposed to cytochalasin D (CD), which is known to disrupt microfilaments. Untreated age-matched embryos were used as controls. Sections of treated embryos and controls were stained with phalloidin, which binds to actin, and examined with a fluorescence microscope. Microdissected specimens were examined using a scanning electron microscope (SEM). Immunofluorescent staining showed a bright signal belt toward the apical cell region of the foregut epithelium in controls. This signal was not evident in CD-exposed embryos. SEM micrographs of the controls showed the cranial foregut as a smooth, even, cylindrical structure in all stages studied. The lumen was narrow and perfectly straight, the ventral and dorsal walls were in close apposition. The foregut of CD-exposed specimens, however, showed a wide lumen and the walls were separated from each other. The structure seemed atonic and appeared conical, curved, or tilted. We observed a dense microfilament network toward the apical cell pole of the epithelial foregut cells of controls that was no longer evident after CD exposure. This network seems to play an important role in foregut morphogenesis, since actin-filament disruption by CD causes loss of the normal shape.

Modulation of inositol 1,4,5-triphosphate concentration by prolyl endopeptidase inhibition.

Prolyl endopeptidase (PEP) is a proline-specific oligopeptidase with a reported effect on learning and memory in different rat model systems. Using the astroglioma cell line U343, PEP expression was reduced by an antisense technique. Measuring different second-messenger concentrations revealed an inverse correlation between inositol 1,4,5-triphosphate [Ins(1,4,5)P3] concentration and PEP expression in the generated antisense cell lines. However, no effect on cAMP generation was observed. In addition, complete suppression of PEP activity by the specific inhibitor, Fmoc-Ala-Pyrr-CN (5 micro m) induced in U343 and other cell lines an enhanced, but delayed, increase in Ins(1,4,5)P3 concentration. This indicates that the proteolytic activity of PEP is responsible for the observed effect. Furthermore, the reduced PEP activity was found to amplify Substance P-mediated stimulation of Ins(1,4,5)P3. The effect of reduced PEP activity on second-messenger concentration indicates a novel intracellular function of this peptidase, which may have an impact on the reported cognitive enhancements due to PEP inhibition.

Hyperosmotic stress induces phosphorylation of cytosolic phospholipase A(2) in HaCaT cells by an epidermal growth factor receptor-mediated process.

Cytosolic phospholipase A(2) (cPLA(2)) is an enzyme involved in the formation of proinflammatory mediators by catalyzing the release of arachidonic acid, thereby mediating eicosanoid biosynthesis. Using HaCaT keratinocytes as a model system, we present experimental evidence that in these cells, cPLA(2) is constitutively phosphorylated and that the degree of phosphorylation dramatically increases in cells under hyperosmotic stress induced by sorbitol. In parallel, a rapid release of arachidonic acid followed by prostaglandin E(2) formation was detected. Elucidating the mechanism of cPLA(2) upregulation, we observed that it is mediated via epidermal growth factor receptor (EGFR) activation, since tyrphostin AG1478, a selective inhibitor of EGFR tyrosine kinase, completely inhibited cPLA(2) phosphorylation. Furthermore, addition of PD98059, which is an inhibitor of MEK1 activation, but not of SB203580, which is an inhibitor of p38 stress kinase, inhibited cPLA(2) phosphorylation, indicating that the ras-raf-MEK cascade is the major signalling pathway involved in cPLA(2) phosphorylation. In addition, depletion of the cells from intracellular calcium does not prevent sorbitol-elicited cPLA(2) phosphorylation, suggesting that this process is independent of the presence of calcium. Together, our results demonstrate that hyperosmotic stress phosphorylates cPLA(2) in human keratinocytes by an EGFR-mediated process.

Ionomycin-activated calpain triggers apoptosis. A probable role for Bcl-2 family members.

Ubiquitous calpains (mu- and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitro both ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-x(L) at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. We conclude that ionomycin-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway.