Tomohiro Banno

A Second Mutant Allele of Furin in the Processing-incompetent Cell Line, LoVo EVIDENCE FOR INVOLVEMENT OF THE HOMO B DOMAIN IN AUTOCATALYTIC ACTIVATION

Furin is a Golgi membrane-associated endoprotease that is involved in cleavage of various precursor proteins predominantly at Arg-X-Lys/Arg-Arg sites. Furin itself is synthesized as an inactive precursor, which is activated through intramolecular autocatalytic cleavage at an Arg-X-Lys-Arg site. We previously found that human colon carcinoma LoVo cells have a frameshift mutation within the homo B domain of furin and thereby lack processing activity toward Arg-X-Lys/Arg-Arg sites. In this study, however, we identified a second furin mutation in this cell line. The mutation, a replacement of a conserved Trp residue within the homo B domain with Arg, results in lack of processing activity of the mutant furin. The combination of both mutations can account for the recessive nature of the processing incompetence of LoVo cells. Immunofluorescence analysis with three distinct anti-furin monoclonal antibodies revealed that neither furin mutant underwent the autocatalytic activation or left the endoplasmic reticulum for the Golgi. These data indicate that the homo B domain as well as the catalytic domain is required for autocatalytic activation of furin.

Pathway-specific Profiling Identifies the NF-κB-dependent Tumor Necrosis Factor α-regulated Genes in Epidermal Keratinocytes

Identification of tumor necrosis factor α (TNFα) as the key agent in inflammatory disorders led to new therapies specifically targeting TNFα and avoiding many side effects of earlier anti-inflammatory drugs. However, because of the wide spectrum of systems affected by TNFα, drugs targeting TNFα have a potential risk of delaying wound healing, secondary infections, and cancer. Indeed, increased risks of tuberculosis and carcinogenesis have been reported as side effects after anti-TNFα therapy. TNFα regulates many processes (e.g. immune response, cell cycle, and apoptosis) through several signal transduction pathways that convey the TNFα signals to the nucleus. Hypothesizing that specific TNFα-dependent pathways control specific processes and that inhibition of a specific pathway may yield even more precisely targeted therapies, we used oligonucleotide microarrays and parthenolide, an NF-κB-specific inhibitor, to identify the NF-κB-dependent set of the TNFα-regulated genes in human epidermal keratinocytes. Expression of ∼40% of all TNFα-regulated genes depends on NF-κB; 17% are regulated early (1–4 h post-treatment), and 23% are regulated late (24–48 h). Cytokines and apoptosis-related and cornification proteins belong to the “early” NF-κB-dependent group, and antigen presentation proteins belong to the “late” group, whereas most cell cycle, RNA-processing, and metabolic enzymes are not NF-κB-dependent. Therefore, inflammation, immunomodulation, apoptosis, and differentiation are on the NF-κB pathway, and cell cycle, metabolism, and RNA processing are not. Most early genes contain consensus NF-κB binding sites in their promoter DNA and are, presumably, directly regulated by NF-κB, except, curiously, the cornification markers. Using siRNA silencing, we identified cFLIP/CFLAR as an essential NF-κB-dependent antiapoptotic gene. The results confirm our hypothesis, suggesting that inhibiting a specific TNFα-dependent signaling pathway may inhibit a specific TNFα-regulated process, leaving others unaffected. This could lead to more specific anti-inflammatory agents that are both more effective and safer.

Transcriptional responses of human epidermal keratinocytes to cytokine interleukin-1

Interleukin‐1 is a proinflammatory and immunomodulatory cytokine that plays a crucial role in inflammatory diseases of the skin, including bacterial infections, bullous diseases, UV damage, and especially psoriasis. To characterize the molecular effects of IL‐1 in epidermis, we defined the transcriptional changes in human epidermal keratinocytes 1, 4, 24, and 48 h after treatment with IL‐1α. IL‐1 significantly regulated 388 genes, including genes associated with proteolysis, adhesion, signal transduction, proliferation, and epidermal differentiation. IL‐1 induces many genes that have antimicrobial function. Secreted cytokines, chemokines, growth factors, and their receptors are the prominent targets of IL‐1 regulation, including IL‐8, IL‐19, elafin, C3, and S100A proteins, which implicate IL‐1 in the pathogenesis of inflammatory diseases. IL‐1 induced not only proliferation‐associated genes but also differentiation marker genes such as transglutaminase‐1 and involucrin, which suggests that IL‐1 plays an important role in the aberrant proliferation and differentiation seen in psoriasis. Correlation of IL‐1 regulated genes with the TNFα and IFNγ regulated ones showed more similarities between IL‐1 and TNFα than IL‐1 and IFNγ, whereas Oncostatin‐M (OsM) affected a largely unrelated set of genes. IL‐1 regulates many genes previously shown to be specifically over‐expressed in psoriasis. In summary, IL‐1 regulates a characteristic set of genes that define its specific contribution to inflammation and aberrant differentiation in skin diseases. J. Cell. Physiol. 214:1–13, 2008.

Inhibition of JNK Promotes Differentiation of Epidermal Keratinocytes

In inflamed tissue, normal signal transduction pathways are altered by extracellular signals. For example, the JNK pathway is activated in psoriatic skin, which makes it an attractive target for treatment. To define comprehensively the JNK-regulated genes in human epidermal keratinocytes, we compared the transcriptional profiles of control and JNK inhibitor-treated keratinocytes, using DNA microarrays. We identified the differentially expressed genes 1, 4, 24, and 48 h after the treatment with SP600125. Surprisingly, the inhibition of JNK in keratinocyte cultures in vitro induces virtually all aspects of epidermal differentiation in vivo: transcription of cornification markers, inhibition of motility, withdrawal from the cell cycle, stratification, and even production of cornified envelopes. The inhibition of JNK also induces the production of enzymes of lipid and steroid metabolism, proteins of the diacylglycerol and inositol phosphate pathways, mitochondrial proteins, histones, and DNA repair enzymes, which have not been associated with differentiation previously. Simultaneously, basal cell markers, including integrins, hemidesmosome and extracellular matrix components, are suppressed. Promoter analysis of regulated genes finds that the binding sites for the forkhead family of transcription factors are over-represented in the SP600125-induced genes and c-Fos sites in the suppressed genes. The JNK-induced proliferation appears to be secondary to inhibition of differentiation. The results indicate that the inhibition of JNK in epidermal keratinocytes is sufficient to initiate their differentiation program and suggest that augmenting JNK activity could be used to delay cornification and enhance wound healing, whereas attenuating it could be a differentiation therapy-based approach for treating psoriasis.

Conformational changes of the smooth endoplasmic reticulum are facilitated by L‐glutamate and its receptors in rat Purkinje cells

The intraventricular administrations of L‐glutamate or trans‐1‐amino‐1,3‐cyclopentanedicarboxylic acid (t‐ACPD), which is an agonist for the metabotropic glutamate receptor, induced conformational changes of the smooth endoplasmic reticulum (SER) to form lamellar bodies, consisting of stacks of flattened cisterns in Purkinje cell dendrites of the rat cerebellum. The formation of lamellar bodies by t‐ACPD or by anoxia was blocked by pretreatment of L(+)‐2‐amino‐3‐phosphonopropionic acid (L‐AP3), which is an antagonist for the metabotropic glutamate receptor. Injections of N‐methyl‐D‐aspartic acid (NMDA) and amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)/kainate, which are categorized as acting on ionotropic receptors of glutamate, did not cause the formation of lamellar bodies, although kainate condensed the dendritic cytoplasm and produced a swelling of surrounding astrocytes. The cisterns of lamellar bodies formed by t‐ACPD were long and formed regular stacks. Many intercisternal bridges were arranged with a center‐to‐center distance of about 25 nm between apposed cisterns. The bridges appeared as short tubes about 15 nm in diameter and in length, a clear center of which linked the lumen of their cisterns. The present results revealed that an excess release of excitatory transmitter by brief anoxia activates metabotropic glutamate receptors, which transform the networks of SER that normally release Ca2+ widely to the neuronal cytoplasm into lamellar bodies. Large Ca2+ storage pools of lamellar bodies are formed by the association of opposing molecules that belong to different cisterns and may protect excess release of Ca2+ from their reservoirs. J. Comp. Neurol. 402:252–263, 1998.

Conformational changes of smooth endoplasmic reticulum induced by brief anoxia in rat Purkinje cells

Morphological changes of the smooth endoplasmic reticulum (SER) in rat cerebellar Purkinje cell dendrites were examined under apneic conditions for 1–5 minutes, induced by an incision of the diaphragm and the collapse of the lungs. The dendrites obtained from control rats contained a tubular network of the SER and hypolemmal cisterns adjacent to the plasma membrane. After a 3–5 minute apnea, the cytoplasm was occupied by many flattened cisterns stacked into lamellae, referred to as “lamellar bodies.” A quantitative analysis revealed that the number of lamellar bodies became maximum after 3 minutes of apnea. After the treatment time, they increased in size by adding new cisterns to the previous core lamellae. This analysis also showed that the total amount of the SER membranes contained in a dendrite did not change during anoxia. Conformational changes from the tubular or hypolemmal SER to lamellar bodies during brief anoxia might occur through a transient and intermediate form of “fenestrated cisterns,” flat across the transverse plane and penetrated by many longitudinally arranged microtubules. We suggest that these morphological changes of the SER during brief anoxia are not fixation artifacts but represent a biological reaction for protecting against intracellular abnormalities during anoxia.

Epidermal cell necrosis with direct epidermal infiltration of Epstein-Barr virus (EBV)-encoded small nuclear RNA-positive T lymphocytes in a patient with EBV-associated haemophagocytic syndrome.

with cryoglobulinaemia is effective and safe. There are two known mechanisms by which cryoglobulins can result in disease. The first is by precipitation within the vascular lumen, typically cold induced, with hyaline plug formation and minimal early-phase inflammation. Typical clinical lesions would be minimally inflammatory cutaneous infarction with or without associated livedo reticularis, or noninflammatory retiform purpura. The second mechanism is that of immune complex vasculitis. As nearly all type II (monoclonal component, usually IgM; polyclonal component, usually IgG) and III (one or more polyclonal components) cryoglobulins are immune complexes, they should all be capable of inducing an immune complex vasculitis, although many do not. Histological examination of an early purpuric lesion in our patient clearly showed cryoglobulin thrombus formation within the dermal vessels. The scarcity of polymorphonuclear leucocytes and leucocytoclasia among the histopathological findings argue against a primary vasculitic process. On the other hand, histology of a recurrent painful nodule revealed leucocytoclastic vasculitis. This is considered to be a secondary event rather than the primary event involved in the pathogenesis. These features favour a thrombo-occlusive vasculopathy rather than a vasculitis per se. Cryoglobulins have been implicated in intravascular thrombotic processes with secondary leucocytoclastic vasculitis that can result in skin infarction and necrosis. In conclusion, we believe that lipo-PGE1 treatment combined with low-dose oral corticosteroid effectively eliminated cryoglobulins and improved leg purpura and skin ulcers in our patient with livedoid vasculitis. Further studies will be needed to clarify the clinical application of lipo-PGE1, but we expect that lipo-PGE1 treatment will be effective among selected patients with cryoglobulinaemic vasculitis.

Keratinocyte Detachment-Differentiation Connection Revisited, or Anoikis-Pityriasi Nexus Redux

Epidermis, a continuously self-renewing and differentiating organ, produces a protective stratum corneum that shields us from external chemical, physical and microbial threats. Epidermal differentiation is a multi-step process regulated by influences, some unknown, others insufficiently explored. Detachment of keratinocytes from the basement membrane is one such pro-differentiation stimulus. Here, we define the transcriptional changes during differentiation, especially those caused by detachment from the substratum. Using comprehensive transcriptional profiling, we revisited the effects of detachment as a differentiation signal to keratinocytes. We identified the genes regulated by detachment, the corresponding ontological categories and, using metaanalysis, compared the genes and categories to those regulated by other pro-differentiating stimuli. We identified 762 genes overexpressed in suspended keratinocyte, including known and novel differentiation markers, and 1427 in attached cells, including basal layer markers. Detachment induced epidermis development, cornification and desmosomal genes, but also innate immunity, proliferation inhibitors, transcription regulators and MAPKs; conversely the attached cells overexpressed cell cycle, anchoring, motility, splicing and mitochondrial genes, and both positive and negative regulators of apoptosis. Metaanalysis identified which detachment-regulated categories overlap with those induced by suprabasal location in vivo, by reaching confluency in vitro, and by inhibition of JUN kinases. Attached and in vivo basal cells shared overexpression of mitochondrial components. Interestingly, melanosome trafficking components were also overexpressed in the attached and in vivo basal keratinocytes. These results suggest that specific pro-differentiation signals induce specific features of the keratinization process, which are in vivo orchestrated into harmonious epidermal homeostasis.

Psoriasis vulgaris and acute guttate psoriasis in a family

A 33‐year‐old man (Case 1) developed scaling and erythema on the extensor side of his extremities, head, and back, 3u2003years before his first visit to our hospital. The lesions spread out to form several plaques and became persistent ( Fig.u20031a ). Serum antistreptolysin O (ASO) was within the normal range and a pharyngeal culture showed normal flora. Histologic findings were compatible with psoriasis vulgaris ( Fig.u20031b ). Topical steroid therapy was effective; however, scaling and erythema persisted.

A case of adult T-cell leukemia/lymphoma with an indolent clinical course has an unusual proviral DNA integration pattern.

Sir, Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell malignancy caused by human T-cell lymphotrophic virus type I (HTLV-I). The characteristic clinical features of ATL include generalized lymphadenopathy, leukemic cells of T-cell origin with highly convoluted nuclei, skin involvement, hypercalcemia and rapid progress of the clinical course. Relatively high rates of HTLV-I infection are observed in parts of Japan, the Caribbean, South America and Africa (1). The transmission of HTLV-I is mainly from mother to child through breast-feeding (2). The subsequent integration of the HTLV-I provirus into T cells is thought to be a random event that occurs during the carrier state. Polyclonal integration patterns of HTLV-I proviral DNA have been demonstrated by Southern blot analysis as the so-called smear pattern in asymptomatic HTLV-I carriers. Of these randomly infected cells, a cell clone is thought to be selected for preferential growth. Although the mechanism of clonal selection remains unclear, the HTLV-I proviral DNA integration pattern can progress from undetectable to polyclonal, or to monoclonal (1). Recently, we encountered a patient with ATL with an indolent clinical course.