Calcium-dependent peripheral localization of 4.1-like proteins and fodrin in cultured human keratinocytes

Summary— Recently, several proteins immunologically related to erythrocyte membrane skeletal proteins, such as protein 4.1 and fodrin (non-erythroid spectrin), have been found in keratinocytes. In the present study, in order to investigate the roles of these proteins in cell-cell contact, we analyzed the distribution of non-erythroid protein 4.1, β-fodrin and actin in cultured human keratinocytes at low (0.15 mM) and standard (1.85 mM) Ca²⁺ concentrations. Immunofluorescence microscopy revealed that immunoreactive forms of protein 4.1, β-fodrin and actin filaments were present in the cytoplasm of cells cultured in low Ca²⁺ medium, while in cells in the standard Ca²⁺ medium, these proteins were localized at the cell boundary and partially in the cytoplasm. When cells in the low-Ca²⁺ medium were treated with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 1 h, these proteins were also present at the cell boundary. Increasing extracellular Ca²⁺ concentration from low to standard in the medium induces cell-cell contact among the cultured human keratinocytes, accompanied by the translocation of protein 4.1 and β-fodrin from the cytoplasm to the membrane. On the basis of the present study, movement of membrane skeletal proteins from the cytosol to the membrane suggests that either these proteins or the membrane skeletal lattice plays an important role in the regulation of cell-cell intergigitations in response to changes in the Ca²⁺ concentrations in culture medium, and that phosphorylation of these skeletal proteins might be involved in the regulation of the membrane skeletal proteins of keratinocytes in response to Ca²⁺.     

Distribution of Calcium and Boron in the Pectin Fraction of Tomato Leaf Cell Wall

Calcium was present in the pectin fraction of tomato leaf cellwall in association with pectin constituents and with a pectin-proteincomplex. Boron deficiency induced a decrease in the amount ofCa associated with pectin constituents. Most of the boron inthe pectin fraction obtained by pectinase treatment was in afree form. These results suggest that boron plays an importantrole in Ca metabolism in the cell wall. ¹Present address: Institute of Applied Biochemistry, Yagi MemorialPark, Mitake, Gifu, Japan. (Received July 31, 1985; Accepted February 18, 1986)     

Synthesis and conformational study of peptides possessing helically arranged ΔZPhe side chains

Z-Dehydrophenylalanine (Δ^zPhe) possessing four oligopeptides, Boc-(L -Ala-Δ^zPhe-Aib)_n-OCH₃ (n = 1–4: Boc, t-butoxycarbonyl; Aib, α-aminoisobutyric acid), were synthesized, and their solution conformations were investigated by ¹H-nmr, ir, uv, and CD spectroscopy and theoretical CD calculation. ¹H-nmr (the solvent accessibility of NH groups) and ir studies indicated that all the NH groups except for those belonging to the N-terminal L -Ala-Δ^zPhe moiety participate in intramolecular hydrogen bonding in chloroform. This suggests that the peptides n = 2–4 have a 4 → 1 hydrogen-bonding pattern characteristic of 3₁₀-helical structures. The uv spectra of all these peptides recorded in chloroform and in trimethyl phosphate showed an intense maximum around 276 nm assigned to the Δ^zPhe chromophores. The corresponding CD spectra of the peptides n = 2–4 showed exciton couplets with a negative peak at longer wavelengths, whereas that of the peptide n = 1 showed only weak signals. Theoretical CD spectra were calculated for the peptides n = 2–4 of several helical conformations, on the basis of exciton chirality method. This calculation indicated that the three peptides form a helical conformation deviating from the perfect 3₁₀-helix that contains three residues per turn, and that their side chains of Δ^z Phe residues are arranged regularly along the helix. The center-to-center distance between the nearest phenyl pair(s) was estimated to be ～ 5.5 Å. The chemical shifts of the Δ^zPhe side-chain protons (H^β and aromatic H) for the peptides n = 2–4 indicated anisotropic shielding effect of neighboring phenyl group(s); the effect also supports a regular arrangement of the Δ^z Phe side chains along the helical axis. © 1993 John Wiley & Sons, Inc.     

Suppression of endoplasmic reticulum stress-induced caspase activation and cell death by the overexpression of Bcl-xL or Bcl-2

Continuous endoplasmic reticulum (ER) stress, such as the accumulation of unfolded proteins, results in cell death and relates to the pathogenesis of some neurodegenerative diseases. Treatment of brefeldin A, an inhibitor of transport between the ER and Golgi complex, induced cell death during 24 h, which accompanied activation of caspase-2, caspase-3 and caspase-9, starting at 12 h and increasing time-dependently up to 28 h. Caspase-2 was expressed and activated in not only mitochondria and cytosol, but also in the microsomal fraction containing ER and Golgi. Of note is that overexpression of Bcl-x(L) or Bcl-2 in PC12 cells markedly suppressed brefeldin A-induced activation of caspases and resulting cell death. Delivery of anti-Bcl-2 antibody into the Bcl-2-overexpressed cells again recovered apoptosis. While the brefeldin A-treatment induced the phosphorylation of both c-Jun N-terminal kinase (JNK) and p38 MAPK, overexpression of Bcl-x(L) or Bcl-2 reduced the prolonged phosphorylation of JNK, but not of p38 MAPK. Pretreatment with a JNK inhibitor, SP600125, suppressed the brefeldin A-induced caspase-2 activation and cell death significantly. Thus, our results suggest that protective effects of Bcl-x(L) and Bcl-2 against brefeldin A-induced cell death appear to be dependent on the regulation of JNK activation.     

Ultraviolet A-induced production of matrix metalloproteinase-1 is mediated by macrophage migration inhibitory factor (MIF) in human dermal fibroblasts

Matrix metalloproteinases (MMPs) are thought to be responsible for dermal photoaging in human skin. In the present study, we evaluated the involvement of macrophage migration inhibitory factor (MIF) in MMP-1 expression under ultraviolet A (UVA) irradiation in cultured human dermal fibroblasts. UVA (20 J/cm(2)) up-regulates MIF production, and UVA-induced MMP-1 mRNA production is inhibited by an anti-MIF antibody. MIF (100 ng/ml) was shown to induce MMP-1 in cultured human dermal fibroblasts. We found that MIF (100 ng/ml) enhanced MMP-1 activity in cultured fibroblasts assessed by zymography. Moreover, we observed that fibroblasts obtained from MIF-deficient mice were much less sensitive to UVA regarding MMP-13 expression than those from wild-type BALB/c mice. Furthermore, after UVA irradiation (10 J/cm(2)), dermal fibroblasts of MIF-deficient mice produced significantly decreased levels of MMP-13 compared with fibroblasts of wild-type mice. Next we investigated the signal transduction pathway of MIF. The up-regulation of MMP-1 mRNA by MIF stimulation was found to be inhibited by a PKC inhibitor (GF109203X), a Src-family tyrosine kinase inhibitor (herbimycin A), a tyrosine kinase inhibitor (genistein), a PKA inhibitor (H89), a MEK inhibitor (PD98089), and a JNK inhibitor (SP600125). In contrast, the p38 inhibitor (SB203580) was found to have little effect on expression of MMP-1 mRNA. We found that PKC-pan, PKC alpha/beta II, PKC delta (Thr505), PKC delta (Ser(643)), Raf, and MAPK were phosphorylated by MIF. Moreover, we demonstrated that phosphorylation of PKC alpha/beta II and MAPK in response to MIF was suppressed by genistein, and herbimycin A as well as by transfection of the plasmid of C-terminal Src kinase. The DNA binding activity of AP-1 was significantly up-regulated 2 h after MIF stimulation. Taken together, these results suggest that MIF is involved in the up-regulation of UVA-induced MMP-1 in dermal fibroblasts through PKC-, PKA-, Src family tyrosine kinase-, MAPK-, c-Jun-, and AP-1-dependent pathways.     

Sister Chromatid Exchanges Are Mediated by Homologous Recombination in Vertebrate Cells

Sister chromatid exchange (SCE) frequency is a commonly used index of chromosomal stability in response to environmental or genetic mutagens. However, the mechanism generating cytologically detectable SCEs and, therefore, their prognostic value for chromosomal stability in mitotic cells remain unclear. We examined the role of the highly conserved homologous recombination (HR) pathway in SCE by measuring SCE levels in HR-defective vertebrate cells. Spontaneous and mitomycin C-induced SCE levels were significantly reduced for chicken DT40 B cells lacking the key HR genes RAD51 and RAD54 but not for nonhomologous DNA end-joining (NHEJ)-defective KU70(-/-) cells. As measured by targeted integration efficiency, reconstitution of HR activity by expression of a human RAD51 transgene restored SCE levels to normal, confirming that HR is the mechanism responsible for SCE. Our findings show that HR uses the nascent sister chromatid to repair potentially lethal DNA lesions accompanying replication, which might explain the lethality or tumorigenic potential associated with defects in HR or HR-associated proteins.