Cutaneous vasculitis and glomerulonephritis associated with C4 deficiency.

Complete deficiency of the fourth component of complement (C4) is an extremely rare condition. However, it has been reported that partial C4 deficiency can occur in normal subjects, and is associated with several immune diseases. We report a 44-year-old woman who developed slight oedema and punctate purpura on her lower legs after a common cold. She was noted to have persistent microscopic haematuria and proteinuria, and her C4 level was undetectable. On histological examination of a skin biopsy specimen, leucocytoclastic vasculitis was seen, with granular deposition of IgG, IgM, C3 and C1q on the vessel walls in the upper dermis. A renal biopsy showed mild mesangial proliferative glomerulonephritis with slight damage to the capillary loops, and granular deposits of IgM and C4 mainly in the mesangium. The patient was systemically well and needed no medication. The C4 level remained low during the observation period, but neither genotyping nor allotyping analysis identified a C4 deficiency.

Revascularization by percutaneous transluminal angioplasty improved abruptly deteriorated ischaemic symptoms in cutaneous polyarteritis nodosa.

We report a patient with cutaneous polyarteritis nodosa, who had a 3-year history of recurrent leg and foot ulcers. Symptoms of ischaemia in the left foot, including severe pain, coldness, paraesthesia and violaceous discoloration, deteriorated abruptly, because of complete occlusion of the left anterior tibial artery. The occluded segment was revascularized by percutaneous transluminal angioplasty, resulting in a dramatic improvement in the ischaemic symptoms.

A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle.

A new protein kinase C (PKC)-related cDNA with unique tissue distribution has been isolated and characterized. This cDNA encodes a protein, nPKC theta, which consists of 707 amino acid residues and showed the highest sequence similarity to nPKC delta (67.0% in total). nPKC theta has a zinc-finger-like cysteine-rich sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are common in all PKC family members. However, nPKC theta lacks a putative Ca2+ binding region (C2 region) that is seen only in the conventional PKC subfamily (cPKC alpha, -beta I, -beta II, and -gamma) but not in the novel PKC subfamily (nPKC delta, -epsilon, -zeta, and -eta). Northern (RNA) blot analyses revealed that the mRNA for nPKC theta is expressed predominantly in skeletal muscle. Furthermore, nPKC theta mRNA is the most abundantly expressed PKC isoform in skeletal muscle among the nine PKC family members. nPKC theta expressed in COS1 cells serves as a phorbol ester receptor. By the use of an antipeptide antibody specific to the D2-D3 region of the nPKC theta sequence, nPKC theta was recognized as a 79-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in mouse skeletal muscle extract and also in an extract from COS1 cells transfected with an nPKC theta cDNA expression plasmid. Autophosphorylation of immunoprecipitated nPKC theta was observed; it was enhanced by phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate but attenuated by the addition of Ca2+. These results clearly demonstrate that nPKC theta should be considered a member of the PKC family of proteins that play crucial roles in the signal transduction pathway.

The eta isoform of protein kinase C is localized on rough endoplasmic reticulum.

The eta isoform of protein kinase C, isolated from a cDNA library of mouse skin, has unique tissue and cellular distributions. It is predominantly expressed in epithelia of the skin, digestive tract, and respiratory tract in close association with epithelial differentiation. We report here that this isoform is localized on the rough endoplasmic reticulum in transiently expressing COS1 cells and constitutively expressing keratinocytes. By the use of polyclonal antibodies raised against peptides of the diverse D1 and D2/D3 regions, we found that immunofluorescent signals were strongest in the cytoplasm around the nucleus and became weaker toward the peripheral cytoplasm. Under immunoelectron microscopic examination, electron-dense signals were located on the rough endoplasmic reticulum and on the outer nuclear membrane which is continuous with the endoplasmic reticulum membrane. However, no signals were detected in the nucleus, inner nuclear membrane, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, or plasma membrane. Treatment of the cells in situ with detergents suggested association of the isoform of protein kinase C with intracellular structures. By immunoblotting, a distinct single band with an M(r) of 80,000 was detected in whole-cell lysate and in rough microsomal and crude nuclear fractions, all of which contain outer nuclear membrane and/or rough endoplasmic reticulum. We further demonstrated the absence of a nuclear localization signal in the pseudosubstrate sequence. The present observation is not consistent with the report of Greif et al. (H. Greif, J. Ben-Chaim, T. Shimon, E. Bechor, H. Eldar, and E. Livneh, Mol. Cell. Biol. 12:1304-1311, 1992).

Urinary liver-type fatty acid-binding protein levels for differential diagnosis of idiopathic focal glomerulosclerosis and minor glomerular abnormalities and effect of low-density lipoprotein apheresis.

AIMS: Focal glomerulosclerosis (FGS) and minor glomerular abnormalities are kidney diseases characterized by massive proteinuria. Urinary liver-type fatty acid-binding protein (L-FABP), an intracellular carrier protein of free fatty acids, is expressed in proximal tubules of the human kidney. Patients with FGS show significant improvement with low-density lipoprotein (LDL) apheresis. The aim of the present study was to determine whether urinary L-FABP levels differ between patients with FGS and those with minor glomerular abnormalities and whether levels are altered by LDL apheresis. PATIENTS AND METHODS: There were 24 patients with minor glomerular abnormalities (nephrotic stage, n = 14, remission stage, n = 10), 17 patients with FGS, and 20 healthy age-matched subjects were included in the present study. Urinary L-FABP levels were measured by enzyme-linked immunosorbent assay and compared. All patients with minor glomerular abnormalities at the nephrotic stage received prednisolone for 6 months, and all FGS patients received some form of immunosuppression therapy with prednisolone, cyclophosphamide or mizoribine for 12 months. LDL apheresis was performed in eight FGS patients with drug-resistant nephrotic syndrome. RESULTS: Urinary L-FABP levels were significantly higher in the 17 FGS patients (82.0 +/- 44.4 microg/g.Cr) than in the 24 patients with minor glomerular abnormalities (10.2 +/- 8.4 microg/g.Cr) (p < 0.01) and in the 20 healthy subjects (7.4 +/- 4.2 microg/g.Cr) (p < 0.01). Urinary L-FABP levels differed little between nephrotic stage and remission stage in patients with minor glomerular abnormalities. Urinary L-FABP levels were significantly higher in the eight drug-resistant FGS patients (122.6 +/- 78.4 microg/g.Cr) than in the nine drug-sensitive FGS patients (45.9 +/- 32.0 microg/g.Cr). Urinary L-FABP levels did not correlate with levels of other clinical markers including serum creatinine, urinary protein, and urinary N-acetyl-beta-D- glucosaminidase. In the eight drug-resistant FGS patients, LDL-apheresis significantly reduced urinary protein excretion (p < 0.01) and urinary L-FABP levels (p < 0.01). CONCLUSIONS: Urinary L-FABP may be a useful diagnostic indicator for differentiation between FGS and minor glomerular abnormalities. LDL apheresis may be effective in ameliorating tubulointerstitial lesions associated with FGS.

Differential modulation of protein kinase C isoforms in erythroleukemia during induced differentiation.

Induction of erythroid differentiation of murine erythroleukemia cells (MELC) by exposure to hexamethylene bisacetamide (HMBA) involves the modulation of protein kinase C (PKC) activity. Using immuno- and Northern blot techniques, we have demonstrated that MELC express a pattern of PKC isoforms which includes PKC alpha, PKC delta, PKC epsilon, PKC zeta, and PKC eta. We show that MELC resistant to induction by HMBA express significantly less of the nPKC isoform, PKC delta, and slightly less PKC epsilon. Recovery of HMBA sensitivity is associated with reexpression of PKC delta protein. Upon exposure to HMBA, there is a fall in cytosolic PKC delta and PKC epsilon accompanied by a transient increase in membrane-associated forms of these PKC isoforms. HMBA-resistant MELC fail to display this isoform-specific translocation of PKC. Induction of differentiation is accompanied, over the next 24 h of exposure to HMBA, by a progressive fall in cellular PKC activity, associated with a progressive fall in the cellular content of PKC delta, PKC epsilon, and PKC zeta. These studies suggest that PKC delta, and possibly PKC epsilon and PKC zeta as well, play a role in the pathway of HMBA-mediated terminal cell differentiation of MELC.

Activation of the JNK pathway by distantly related protein kinases, MEKK and MUK.

JNK/SAPKs are identified as new members of the MAPK family; they phosphorylate c-Jun protein in response to several cellular stimuli including ultraviolet irradiation, TNF and osmotic shock. We have identified a protein kinase, MUK, as an activator of the JNK-pathway, whose kinase domain shows significant homology to MAPKKK-related proteins such as c-Raf and MEKK. The over-expression of MUK or MEK kinase (MEKK) in NIH3T3 or COS1 cells results in the activation of JNK1 and the accumulation of a hyper-phosphorylated form of c-Jun. While MEKK also activates the ERK pathway, MUK is a rather selective activator of the JNK pathway. On the other hand, c-Raf activates the JNK pathway only slightly despite its remarkable ability to activate the ERK pathway. Even though we originally identified MUK as a MAPKKK-related protein kinase, a greater similarity to mixed lineage kinase (MLK) is found not only in the catalytic domain but also in the 'leucine-zipper'-like motifs located at the C-terminal side of the catalytic domain. The structural divergence between MUK and MEKK reveals the multiplicity of signaling pathways that activate JNK/SAPKs.

YSK1, a novel mammalian protein kinase structurally related to Ste20 and SPS1, but is not involved in the known MAPK pathways.

To clarify the upstream regulatory mechanism of mitogen-activated protein kinase (MAPK), we performed the reverse transcriptase-based polymerase chain reaction (RT-PCR) with degenerate primers synthesized based on sequences conserved among the kinase domains of yeast MAPK kinase kinases (MAPKKKs), Stell, Bck1, and Byr2. We isolated several mammalian cDNA fragments that encode kinase subdomains sharing significant sequence homology with yeast MAPKKKs. Subsequent screening of a HeLa cell cDNA library using one of these cDNA fragments as a probe resulted in the isolation of a full-length cDNA that encodes a novel protein kinase. The catalytic domain sequence of this gene product is closely related to those of budding yeast Sps1 and Ste20 protein kinases. Thus, we call this protein YSK1 (Yeast Sps1/Ste20-related Kinase 1). The transcript of YSK1 was detected in a wide range of tissues and cells. Immunoprecipitated YSK1 shows protein kinase activity. Although YSK1 is significantly similar in its kinase domain to kinases of the yeast and mammalian MAPK pathways, the overexpression of YSK1 did not lead to the activation of the ERK (extracellular signal-regulated kinase) pathway, JNK (c-Jun NH2-terminal kinase)/SAPK (stress-activated protein kinase) pathway, or p38/Mpk2 pathway. These results suggest that YSK1 may be involved in the regulation of a novel intracellular signaling pathway.

Cytosolic calcium oscillations induced by hepatocyte growth factor (HGF) in single fura-2-loaded cultured hepatocytes: effects of extracellular calcium and protein kinase C.

Hepatocyte growth factor (HGF) induced the periodic fluctuations of cytosolic calcium concentration ([Ca2+]i) in primary cultured rat hepatocytes, which were dependent on extracellular calcium. The HGF-induced [Ca2+]i oscillations were suppressed by the pretreatment with phorbol 12-myristate 13-acetate (PMA). Administration of PMA during oscillations also caused their blockade, but the subsequent addition of protein kinase C (PKC) inhibitor H-7 reversed the inhibitory effects of PMA, thereby resulting in the resumption of the oscillatory responses. Moreover, the prior exposure to H-7 caused apparent increases in [Ca2+]i spike peaks elicited by HGF. These results suggest a negative modulation via PKC in HGF-induced repetitive [Ca2+]i transients. The absence of HGF-induced oscillations after the thapsigargin treatment indicates that the agonist-sensitive intracellular Ca2+ pool plays a crucial role in the [Ca2+] oscillations.

A new thiazolidinedione, NC-2100, which is a weak PPAR-gamma activator, exhibits potent antidiabetic effects and induces uncoupling protein 1 in white adipose tissue of KKAy obese mice.

Thiazolidinediones (TZDs) reduce insulin resistance in type 2 diabetes by increasing peripheral uptake of glucose, and they bind to and activate the transcriptional factor peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Studies have suggested that TZD-induced activation of PPAR-gamma correlates with antidiabetic action, but the mechanism by which the activated PPAR-gamma is involved in reducing insulin resistance is not known. To examine whether activation of PPAR-gamma directly correlates with antidiabetic activities, we compared the effects of 4 TZDs (troglitazone, pioglitazone, BRL-49653, and a new derivative, NC-2100) on the activation of PPAR-gamma in a reporter assay, transcription of the target genes, adipogenesis, plasma glucose and triglyceride levels, and body weight using obese KKAy mice. There were 10- to 30-fold higher concentrations of NC-2100 required for maximal activation of PPAR-gamma in a reporter assay system, and only high concentrations of NC-2100 weakly induced transcription of the PPAR-gamma but not PPAR-alpha target genes in a whole mouse and adipogenesis of cultured 3T3L1 cells, which indicates that NC-2100 is a weak PPAR-gamma activator. However, low concentrations of NC-2100 efficiently lowered plasma glucose levels in KKAy obese mice. These results strongly suggest that TZD-induced activation of PPAR-gamma does not directly correlate with antidiabetic (glucose-lowering) action. Furthermore, NC-2100 caused the smallest body weight increase of the 4 TZDs, which may be partly explained by the finding that NC-2100 efficiently induces uncoupling protein (UCP)-2 mRNA and significantly induces UCP1 mRNA in white adipose tissue (WAT). NC-2100 induced UCP1 efficiently in mesenteric WAT and less efficiently in subcutaneous WAT, although pioglitazone and troglitazone also slightly induced UCP1 only in mesenteric WAT. These characteristics of NC-2100 should be beneficial for humans with limited amounts of brown adipose tissue.

mRNA expression of growth factors in glomeruli from diabetic rats.

Evaluations of glomerular mRNA levels encoding for PCNA, TNF-alpha, PDGF-A and -B chains, TGF-beta, IGF-I, bFGF, and EGF were made at 4, 12, and 24 wk after injection of STZ in Sprague-Dawley rats. The mRNA levels for PCNA, TNF-alpha, PDGF-B chain, TGF-beta, and bFGF increased with age in STZ-induced diabetic rats. At 24 wk after STZ injection, mRNA levels for PCNA, TNF-alpha, PDGF-B chain, TGF-beta, and bFGF were increased 3.8-fold, (P < 0.01), 4.2-fold (P < 0.01), 4.0-fold (P < 0.01), 5.2-fold (P < 0.001), and 3.6-fold (P < 0.01), respectively, in the glomeruli of diabetic rats when compared with control rats. In contrast, mRNA levels for IGF-I, PDGF-A chain, and EGF were not altered in glomeruli from diabetic and control rats throughout the experimental period. Insulin treatment partially ameliorated the increase in mRNA levels for PCNA, TNF-alpha, PDGF-B chain, TGF-beta, and bFGF in the glomeruli of diabetic rats. These data indicate that alterations in growth factor mRNA levels in glomeruli may be a manifestation of diabetic nephropathy, and that hyperglycemia or insulin deficiency may play a role in abnormal growth factor gene regulation.

Tumor cell growth inhibition and extracellular signal-regulated kinase (ERK) phosphorylation by novel K vitamins.

2-(2-hydroxy-ethylsulfanyl)-3-methyl-1,4-naphthoquinone or CPD-5, a K vitamin analog, was previously indicated to be a potent growth inhibitor for Hep 3B hepatoma cells in vitro. Here, we show that CPD-5 and two newly synthesized analogs, 2-(2-hydroxy-ethylsulfanyl)-3-methyl-5- nitro-1,4-naphthoquinone (PD-37) and 2-(2-hydroxy-ethylsulfanyl)-3- methyl-5-acetylamino-1,4-naphthoquinone (PD-42), are potent growth inhibitors of 13 different human cancer cell lines, with IC50 values in the range of 3-54 microM. Phospho-ERK was induced by each of three K vitamin analogs in every cell line in a dose-dependent manner, at growth inhibitory doses. ERK phosphorylation and growth inhibitory effects were strongly correlated, with p=0.0080 for CPD-5, p=0.0076 for PD-37 and p=0.0251 for PD-42. The induction of phospho-ERK and growth inhibition were antagonized by thiol-containing anti-oxidants, but not by catalase, consistent with a possible arylating mechanism. The data show a novel class of growth inhibitors with a wide spectrum of action that induces ERK hyper-phosphorylation, as a possible new growth inhibitory feature.

Differentiation-associated localization of nPKC eta, a Ca(++)-independent protein kinase C, in normal human skin and skin diseases.

The expression of nPKC eta, a Ca(++)-independent isoform of protein kinase C in normal human skin, and skin from patients with psoriasis, squamous cell carcinoma, basal cell epithelioma, nevus pigmentosus, and seborrheic keratosis, were examined by immunohistochemical staining using a polyclonal antibody raised against a synthetic peptide at a diverse region of the nPKC eta molecule. In normal epidermis, the strongest staining was observed in the uppermost granular layer with no staining of the spinous or basal layers. The inner layer of the intra-epidermal eccrine duct was also strongly stained. Weak staining was observed in several layers of the outer root sheath of the follicular infundibulum. No staining was detected in the inner root sheath of the hair follicles, hair matrix, sebaceous gland, eccrine gland, intradermal eccrine duct, arrectores pilorum, melanocytes, Langerhans cells, fibroblasts, or blood vessels. In psoriatic skin, stained keratinocytes were distributed in the suprabasal layers with the most being observed in the uppermost layer and the least in layers closed to the basal layer. In squamous cell carcinoma, weak staining was observed in the keratotic cells around horny pearls. In the basal cell epithelioma and nevus pigmentosus, the cells were not stained, whereas in seborrheic keratosis, cells that stained were located in the granular layer. We conclude from the evidence presented above that nPKC eta is expressed in close association with epidermal differentiation in normal skin and skin diseases.

Sequence and expression patterns of mouse SPR1: Correlation of expression with epithelial function.

A final event in the terminal differentiation of stratified squamous epithelia is the formation of a cornified cell envelope, which is a complex of several proteins cross-linked together by transglutaminases. One set of proteins is the family of small proline rich (SPR) proteins. In human foreskin epidermal cell envelopes, SPRs serve as cross-bridging proteins among the more abundant loricrin. In order to study further their evolution and expression, we have isolated and sequenced cDNAs encoding two mouse SPR1 proteins, SPR1a and SPR1b Comparative sequence analysis showed the preservation of the overall structure of mammalian SPR1 proteins with highly conserved termini and a central peptide domain repeated 13 (SPE1a) or seven (SPR1b) times. Tissues obtained from mouse fetal, newborn, and adult skin were tested by Northern blot analyses, in situ hybridization and immunohistochemistry using an antibody raised to a synthetic peptide corresponding to the C terminus of the SPR1a protein. Skin expression was first detected in fetal periderm in anagen hair follicles of newborn and older mice, and in the thickened epidermis of the lip and footpad, but no signal was detected in interfollicular trunk epidermis. High levels of SPR1a expression were found in epithelia from the forestomach and penis, and in benign squamous papillomas. Other epithelia expressing SPR1a include the tongue, esophagus, and vagina. Whenever detected, SPR1a positive staining was present in the spinous and granular layers. In the forestomach and papillomas, the periphery of cells in the cornified layer was also stained. Our results suggest that SPR1a participates widely in the construction of cell envelopes in cornifying epithelia characterized by either increased thickness or a requirement for extreme flexibility. Based on its likely function as a cross-bridging protein in cell envelopes, we conclude that the mechanical attributes of cell envelopes may be determined in part by the SPR1 content, in accordance with the specific function of the epithelium.

Sequence analysis of the rat jun-D gene.

A rat jun-D genomic clone was isolated from a rat liver lambda EMBL3 library. Sequence analysis revealed a high sequence similarity with mouse jun-D, and relatively low similarity with human and chicken jun-D. In both the trans-activation and DNA-binding domains, however, the amino-acid sequences were well conserved among rat, mouse, human and chicken.

Identification of DNA binding-site preferences for nuclear factor I-A.

Nuclear factor I (NFI) proteins constitute a large family of DNA binding proteins. These proteins promote the initiation of adenovirus replication and regulate the transcription of viral and cellular genes. The binding sites for NFI have been reported in a wide variety of promoters, and they exhibit flexibility in their sequences. To clarify the DNA binding site of NFI-A, one of the NFI proteins, we performed a polymerase chain reaction-mediated random site selection, and determined the optimal sequence as 5'-TTGGCANNNN(G/T)CCA(G/A)-3'.

A domain containing the Cdc42/Rac interactive binding (CRIB) region of p65PAK inhibits transcriptional activation and cell transformation mediated by the Ras-Rac pathway.

The molecular bases of the versatile functions of Rho-like GTPases are still unknown. Using luciferase assays with rat 3Y1 cells, we found that Rac1 is integrated downstream of Ras in the TRE (TPA response element) activation pathway. Coexpression of a mutant of p65PAK, PAK/RD, lacking the kinase domain but containing the Cdc42/Rac interactive binding (CRIB) region, suppressed the TRE activation and cell transformation caused by constitutively activated forms of Ras (RasV12) and Rac1 (Rac1V12). PAK/RD is a good tool to investigate the signaling pathways in which Rac and Cdc42 are involved.

Hepatocyte growth factor (HGF) mediates the sustained formation of 1,2-diacylglycerol via phosphatidylcholine-phospholipase C in cultured rat hepatocytes.

The addition of hepatocyte growth factor (HGF) to rat hepatocytes in primary culture resulted in the formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and 1,2-diacylglycerol (DG) by a phosphoinositide-specific phospholipase C (PI-PLC). DG showed a biphasic increase; the first phase, corresponding with the peak of Ins(1,4,5)P3 and a second larger and prolonged phase. The HGF stimulates the phosphatidylcholine (PC)-derived prolonged DG formation by a phospholipase C pathway (PC-PLC) but not by a phospholipase D pathway. HGF also was found to elicit [Ca2+] oscillations which may be associated with the prolonged DG production from PC via the PC-PLC phospholipase C pathway.