Chromosomal localization of the human interleukin 1α (IL-1α) gene

The human interleukin 1α gene was assigned to chromosome 2 using Southern transfer analysis of human-rodent somatic cell hybrid DNAs. The gene was regionally localized to 2q12–21 using in situ hybridization to metaphase chromosomes. These results indicate that the IL-1α gene maps to the same general region on the long arm of chromosome 2 as the IL-1β gene, which has been previously assigned.     

The α6β4 Integrin Is a Receptor for both Laminin and Kalinin

Previously, we have established K562 transfectants that express either α6Aβ1 or α6Bβ1 (Kα6A or Kα6B) on their surface. Both cell lines bind to laminin and kalinin after treatment with the β1-stimulatory antibody TS2/16. Here we introduce the full-length β4 cDNA into the α6A- and α6B-expressing K562 cells and selected stably transfected cells. The β4 subunit was expressed on the surface of both transfectants and it formed dimers with the α6A or α6B subunits. Immunoprecipitation and preclearing analyses revealed that both transfectants expressed α6β1, in addition to α6β4. While Kα6A and Kβ6B cells required TS2/16 stimulation for binding to laminin or kalinin, adhesion of the unstimulated β4-transfected Kα6A and Kα6B cells to these matrix components was already substantial. This adhesion was mediated by both α6β1 and α6β4 since it was completely blocked by an α6-specific antibody or by a combination of anti-β1 and anti-β4 antibodies, but only partially by either of these latter two antibodies alone. Adhesion to laminin was completely blocked by an antiserum to laminin fragment E8 as was the adhesion to kalinin by an antibody to kalinin, demonstrating the specificity of adhesion. Both transfectants always adhered more strongly to kalinin than to laminin. Furthermore, binding to kalinin was less well blocked by antibodies to β4 than binding to laminin, indicating that the affinity of α6β4 for kalinin is higher than that for laminin. The fact that α6β1 mediated adhesion without TS2/16 stimulation on the β4-transfected Kα6A and Kα6B cells suggests that some activation of α6β1 had occurred in these cells, even though binding was increased when they were actively stimulated by the antibody TS2/16. Finally, we show that Mn²⁺ induced binding of solubilized α6β4 to matrix containing kalinin, deposited by the murine cell line RAC-11P/SD. This binding was inhibited by the anti-α6 mAb GoH3. Together, these results indicate that both α6β1 and α6β4 are receptors for laminin and kalinin and that there are no differences in ligand specificity between the A and B variants of the α6 subunit when associated with either β1 or β4.     

A Lymphocyte Serine Protease Granzyme A Causes Detachment of a Small-Intestinal Epithelial Cell Line (IEC-6)

Granzyme A (GzmA) is a serine protease (trypsin-like specificity) produced in cytotoxic lymphocytes. This enzyme is believed to enter virus-infected cells and growing tumors and induce apoptosis, but the roles of GzmA expressed in lymphocytes scattered through the epithelial layer of the normal small intestine are unknown. In the present study, recombinant rat GzmA (rGzmA) was found to cause morphological changes and detachment of a non-transformed rat small-intestinal epithelial cell line IEC-6, although the rGzmA-treated cells detached as aggregates with no changes characteristic of apoptosis. rGzmA-induced deformation and detachment occurred in IEC-6 cells plated with collagen type IV and fibronectin, but not in those plated with laminin. These findings suggest that GzmA in the normal small intestine participates in the reduction of adhesion between epithelial cells and basement membranes, through its ability to cleave extracellular matrix components.     

Mechanisms involved in α6β1-integrin-mediated Ca signalling

Contact of Jurkat T-lymphocytes with the extracellular matrix (ECM) protein laminin resulted in long-lasting α6β1-integrin-mediated Ca²⁺ signalling. Both Ca²⁺ release from thapsigargin-sensitive Ca²⁺ stores and capacitative Ca²⁺ entry via Ca²⁺ channels sensitive to SKF 96365 constitute important parts of this process. Inhibition of α6β1-integrin-mediated Ca²⁺ signalling by (1) the src kinase inhibitor PP2, (2) the PLC inhibitor U73122, and (3) the cyclic adenosine diphosphoribose (cADPR) antagonist 7-deaza-8-Br-cADPR indicate the involvement of src tyrosine kinases and the Ca²⁺-releasing second messengers d-myo-inositol 1,4,5-trisphosphate (InsP₃) and cADPR.     

α6 Integrin Is Regulated with Lens Cell Differentiation by Linkage to the Cytoskeleton and Isoform Switching

The developing chicken embryo lens provides a unique model for examining the relationship between α6 integrin expression and cell differentiation, since multiple stages of differentiation are expressed concurrently at one stage of development. We demonstrate that α6 integrin is likely to mediate the inductive effects of laminin on lens differentiation as well as to function in a matrix-independent manner along the cell–cell interfaces of the differentiating cortical lens fiber cells. Both α6 isoform expression and its linkage to the cytoskeleton were regulated in a differentiation-specific manner. The association of α6 integrin with the Triton-insoluble cytoskeleton increased as the lens cells differentiated, reaching its highest levels in the cortical fiber region where the lens fiber cells are formed. In this region of the lens α6 integrin was uniquely localized along the cell–cell borders of the differentiating fiber cells, similar to β1. α6β4, the primary transmembrane protein of hemidesmosomes, is also expressed in the lens, but in the absence of hemidesmosomes. Differential expression of α6A and α6B isoforms with lens cell differentiation was seen at both the mRNA and the protein levels. RT-PCR studies demonstrated that α6B was the predominant isoform expressed both early in development, embryonic day 4, and in the epithelial regions of the day 10 embryonic lens. Isoform switching, with α6A now the predominant isoform, occurred in the fiber cell zones. Immunoprecipitation studies showed that α6B, which is characteristic of undifferentiated cells, was expressed by the lens epithelial cells but was dramatically reduced in the lens fiber zones. Expression of α6B began to drop as the cells initiated their differentiation and then dropped precipitously in the cortical fiber zone. In contrast, expression of the α6A isoform remained high until the cells became terminally differentiated. α6A was the predominant isoform expressed in the cortical fiber region. The down-regulation of α6B relative to α6A provides a developmental switch in the process of lens fiber cell differentiation.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

A Cell Signal Pathway Involving Laminin-5, α3β1 Integrin, and Mitogen-activated Protein Kinase Can Regulate Epithelial Cell Proliferation

Laminin-5 (LN5) is a matrix component of epithelial tissue basement membranes and plays an important role in the initiation and maintenance of epithelial cell anchorage to the underlying connective tissue. Here we show that two distinct LN5 function-inhibitory antibodies, both of which bind the globular domain of the α3 subunit, inhibit proliferation of epithelial cells. These same antibodies also induce a decrease in mitogen-activated protein kinase activity. Inhibition of proliferation by the function-perturbing LN5 antibodies is reversed upon removal of the antibodies and can be overcome by providing the antibody-treated cells with exogenous LN5 and rat tail collagen. Because epithelial cells use the integrin receptor α3β1 to interact with both LN5 and rat tail collagen, we next investigated the possibility that integrin α3β1 is involved in mediating the proliferative impact of LN5. Proliferation of human epithelial cells is significantly inhibited by a function-perturbing α3 integrin antibody. In addition, antibody activation of β1 integrin restores the proliferation of epithelial cells treated with LN5 function-perturbing antibodies. These data indicate that a complex comprising LN5 and α3β1 integrin is multifunctional and contributes not only to epithelial cell adhesion but also to the regulation of cell growth via a signaling pathway involving mitogen-activated protein kinase. We discuss our study in light of recent evidence that LN5 expression is up-regulated at the leading tips of tumors, where it may play a role in tumor cell proliferation.     

Mechanical Induction of β1-Integrin-Mediated Calcium Signaling in a Hepatocyte Cell Line

Mechanical stress influences growth, differentiation, and gene expression in a variety of cell types. It is believed that via extracellular matrix the mechanical stimulus is transmitted to integrin receptors which thus play a key role in transducing signals into the cell interior. Here we demonstrate that incubation of suspended hepatocytes with specific antibodies to β1-integrin subunits followed by a short-term mechanical stimulation is sufficient to induce a rise in intracellular Ca²⁺. The results indicate that mechanical loading of individual integrin subunits activates Ca²⁺-specific signal pathways.     

Blood Platelets Contain and Secrete Laminin-8 (α4β1γ1) and Adhere to Laminin-8 via α6β1 Integrin

Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin γ1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to γ1 and β1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin β1 antibody–Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to β1 and γ1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin α4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20–35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to β1 and α6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (α4β1γ1) and that the cells adhere to the protein by using α6β1 integrin.     

Involvement of PKC-β in PTH, TNF-α, and IL-1β Effects on IL-6 Promoter in Osteoblastic Cells and on PTH-Stimulated Bone Resorption

Protein kinase C (PKC) has been shown to be activated by parathyroid hormone (PTH) in osteoblasts. Prior evidence suggests that this activation mediates responses leading to bone resorption, including production of the osteoclastogenic cytokine interleukin-6 (IL-6). However, the importance of specific PKC isozymes in this process has not been investigated. A selective antagonist of PKC-β, LY379196, was used to determine the role of the PKC-β isozyme in the expression of IL-6 in UMR-106 rat osteoblastic cells and in bone resorption in fetal rat limb bone organ cultures. PTH, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) induced translocation of PKC-α and -β_I to the plasma membrane in UMR-106 cells within 5 min. The stimulation of PKC-β_I translocation by PTH, TNF-α or IL-1β was inhibited by LY379196. In contrast, LY379196 did not affect PTH, TNF-α-, or IL-1β-stimulated translocation of PKC-α. PTH, TNF-α, and IL-1β increased luciferase expression in UMR-106 cells transiently transfected with a −224/+11 bp IL-6 promoter-driven reporter construct. The IL-6 responses were also attenuated by treatment with LY379196. Furthermore, LY379196 inhibited bone resorption elicited by PTH in fetal rat bone organ cultures. These results indicate that PKC-β_I is a component of the signaling pathway that mediates PTH-, TNF-α-, and IL-1β-stimulated IL-6 expression and PTH-stimulated bone resorption.     

Structural classification of αββ and ββα supersecondary structure units in proteins

We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α–β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α–β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193–212, 1998. © 1998 Wiley-Liss, Inc.     

Cell density—dependent regulation of PLCγ1 tyrosine phosphorylation and catalytic activity in an intestinal cell line (IEC-6)

Administration of epidermal growth factor (EGF) to rats has been shown to induce both mitogenic and nonmitogenic responses in the intestine. The mechanisms to describe a multiplicity of hormonal responses within a single tissue are unclear but likely involve selectivity among receptor substrates. A nontransformed rat jejunal crypt intestinal epithelial cell line (IEC-6) was studied to determine if the regulation of receptor tyrosine kinase substrates is affected by cell population physiology. EGF stimulated a rapid increase in inositol trisphosphate in confluent but not subconfluent cells. Similarly, treatment of confluent IEC-6 cells with EGF provoked a significant increase in the hydrolysis of Ptdlns 4,5-P₂ by immunoisolated PLCγ1. The tyrosine phosphorlation state of PLCγ1 and the association of PLCγ1 with the EGF receptor were increased by EGF in confluent cells only. In contrast, the autophosphorylation state of the EGF receptor and the tyrosine phosphorylation state of another SH2-containing EGF receptor substrate SHC were increased by EGF regardless of cell density. Western blot analysis revealed equal protein expression of PLCγ1 in confluent and subconfluent cells. EGF receptor protein expression and ligand binding capacity were slightly increased in confluent compared to subconfluent cells. EGF regulation of PLCγ1, therefore, is regulated by physiological factors dependent on cell density in IEC-6 cells. © 1995 Wiley-Liss, Inc.     

β-Turn conformations in crystal structures of model peptides containing α,α-Di-n-propylglycine and α,α-Di-n-butylglycine

The crystal state conformations of three peptides containing the α,α-dialkylated residues. α,α-di-n-propylglycine (Dpg) and α,α-di-n-butylglycine (Dbg), have been established by x-ray diffraction. Boc-Ala-Dpg-Alu-OMe (I) and Boc-Ala-Dbg-Ala-OMe (III) adopt distorted type II β-turn conformations with Ala (1) and Dpg/Dbg (2) as the corner residues. In both peptides the conformational angles at the Dxg residue (I: ? = 66.2°, ψ = 19.3°; III: ? = 66.5°. ψ = 21.1°) deviate appreciably from ideal values for the i + 2 residue in a type II β-turn. In both peptides the observed (N…O) distances between the Boc CO and Ala (3) NH groups are far too long (1: 3.44 Å: III: 3.63 Å) for an intramolecular 4 → 1 hydrogen bond. Boc-Ala-Dpg-Ata-NHMe (II) crystallizes with two independent molecules in the asymmetric unit. Both molecules HA and HB adopt consecutive β-turn (type III-III in HA and type III-I in IIB) or incipient 3₁₀-helical structures, stabilized by two intramolecular 4 → 1 hydrogen bonds. In all four molecules the bond angle N-C^α-C′ (τ) at the Dxg residues are ≥ 110°. The observation of conformational angles in the helical region of ?,ψ space at these residues is consistent with theoretical predictions. © 1995 John Wiley & Sons, Inc.     

Role of the I-domain in collagen binding specificity and activation of the integrins α1β1 and α2β1

Adhesion to collagens by most cell types is mediated by the integrins α1β1 and α2β1. Both integrin α subunits belong to a group which is characterized by the presence of an I domain in the N-terminal half of the molecule, and this domain has been implicated in the ligand recognition. Since purified α1β1 and α2β1 differ in their binding to collagens I and IV and recognize different sites within the major cell binding domain of collagen IV, we investigated the potential role of the α1 and α2 I domains in specific collagen adhesion. We find that introducing the α2 I domain into α1 results in surface expression of a functional collagen receptor. The adhesion mediated by this chimeric receptor (α1-2-1β1) is similar to the adhesion profile conferred by α2β1, not α1β1. The presence of α2 or α1-2-1 results in preferential binding to collagen I, whereas α1 expressing cells bind better to collagen IV. In addition, α1 containing cells bind to low amounts of a tryptic fragment of collagen IV, whereas α2 or α1-2-1 bearing cells adhere only to high concentrations of this substrate. We also find that collagen adhesion of NIH-3T3 mediated by α2β1 or α1-2-1β1, but not by α1, requires the presence of Mn²⁺ ions. This ion requirement was not found in CHO cells, implicating the I domain in cell type-specific activation of integrins. J. Cell. Physiol. 176:634–641, 1998. © 1998 Wiley-Liss, Inc.