Masaru Honma

Unique Keratinization Process in Psoriasis: Late Differentiation Markers Are Abolished Because of the Premature Cell Death

The keratinization process in psoriasis is a unique phenomenon. We have proposed an organized system for keratinization in psoriasis based on the recognition of early and late differentiation markers combined with premature cell death. The early differentiation markers, such as involucrin, small proline‐rich proteins (SPRR), cystatin A and transglutaminase l, are more conspicuously expressed in psoriasis, while the late differentiation markers, such as profilaggrin and loricrin, are abolished. Keratinization markers that are not observed in the normal epidermis are also detected; these include SKALP/elafin as well as K6 and K16. With a markedly diminished turnover time, the psoriatic epidermis rapidly synthesizes differentiation markers that are mostly under the control of the protein kinase C‐AP1 transcriptional control system. Because of the premature cell death, however, the late differentiation markers are not expressed. During the improvement of the lesion and the therefore longer turnover time, the late differentiation markers rapidly catch up to reveal their expression. This explains the rapid appearance of keratohyalin granules (profilaggrin) in the healing lesion of psoriasis. Thus the keratinization process in psoriasis can be explained by the accelerated keratinization combined with premature cell death. The keratinization process in psoriasis is unique, because both accelerated keratinization and premature cell death co‐exist, resulting in the disappearance of late differentiation markers such as profilaggrin and loricrin. It is interesting to note that the premature cell death is also under the control of protein kinase C signaling.

Podoplanin expression in wound and hyperproliferative psoriatic epidermis: Regulation by TGF-β and STAT-3 activating cytokines, IFN-γ, IL-6, and IL-22

BACKGROUND Podoplanin (PDPN)/T1α/aggrus/PA2.26 antigen, a transmembranous glycoprotein, is a well-known lymphatic endothelial marker. Recent evidence indicates that PDPN is also expressed in keratinocytes especially of sebaceous glands. OBJECTIVE To verify expression-pattern and the regulatory mechanism of PDPN in human epidermal keratinocytes. METHODS PDPN-expression pattern was analyzed in normal and psoriatic epidermis by immunostaining. The regulatory mechanism of PDPN-expression of keratinocytes by cytokines was analyzed using specific inhibitors, siRNA, and adenoviral shRNA of signaling pathways. RESULTS In normal skin, PDPN was expressed on the basal cell layer of sebaceous glands and on the outer root sheath of hair follicles. While no expression was detected in the normal interfollicular epidermis, PDPN was detected in the basal cell layer of wound and hyperproliferative psoriatic epidermis, where the granular layer is lacking. TGF-β1 and IFN-γ independently upregulated PDPN-expression of keratinocytes via TGF-β receptor-Smad pathway and JAK-STAT pathway, respectively. IL-6 and IL-22 also stimulated PDPN-expression of keratinocytes accompanied by STAT-3 phosphorylation. siRNA of STAT-1, inhibitors of STAT-3 signaling, AG490, STAT-3 inhibitor VI, and si/shRNA of STAT-3 inhibited the PDPN-expression of keratinocytes induced by IFN-γ, IL-6 and IL-22 but not by TGF-β1. CONCLUSION These results indicate that TGF-β1, IFN-γ, IL-6, and IL-22 induce PDPN-expression of keratinocytes, which might be significantly involved in the wound healing process as well as in the pathomechanism of hyperproliferative psoriatic epidermis.

Tight junctions in the stratum corneum explain spatial differences in corneodesmosome degradation.

Abstract:  To maintain stratum corneum integrity while simultaneously desquamating at a steady rate, degradation of corneodesmosomes must proceed in a controlled manner. It is unknown why corneodesmosomes are present only at the cell periphery in the upper stratum corneum. To explore this, we studied distributions of three major corneodesmosomal components, corneodesmosin, desmoglein 1 and desmocollin 1 in normal adult human epidermis. Immunofluorescent microscopy studies of skin surface corneocytes detected all three components only at the cell edges. Immunoelectron microscopy revealed selective loss of these components at the central areas starting from the deep cornified layers. We hypothesized that tight junctions (TJs) formed in the superficial granular layer may prevent protease access by functioning as a barrier between the peripheral and the central intercellular spaces in the stratum corneum. Ultrastructural examination demonstrated TJs up to the junctions between the seventh and the eighth deepest cornified layers. Immunoelectron microscopy also detected clusters of occludin and claudin‐1 immunolabels at the cell periphery, and kallikrein 7 immunolabels outside of TJs in the lower cornified layers. With colloidal lanthanum nitrate perfusion assay of stripped stratum corneum, the tracer was excluded from TJ domains. Taken together, we propose that TJs inhibit access of proteases to the peripheral corneodesmosomes forming the structural basis for the basket‐weave‐like appearance of the stratum corneum.

Efficacy and safety of secukinumab in patients with generalized pustular psoriasis: A 52-week analysis from phase III open-label multicenter Japanese study.

Generalized pustular psoriasis (GPP) is a severe inflammatory skin disease characterized by the presence of sterile pustules covering almost the entire body and systemic symptoms such as fever. Secukinumab, a fully human‐recombinant anti‐interleukin‐17A monoclonal antibody was indicated for psoriasis vulgaris and psoriatic arthritis in Japan but is not yet investigated for GPP. In this phase III, open‐label multicenter single arm study, the efficacy and safety of secukinumab as monotherapy or with co‐medication was evaluated in 12 Japanese patients with GPP. All the patients received secukinumab 150 mg s.c. at baseline, week 1, 2, 3 and 4, and then every 4 weeks. Two non‐responders were up‐titrated to 300 mg. Change in GPP severity from baseline was evaluated by clinical global impression (CGI) categorized as “worsened”, “no change”, “minimally improved”, “much improved” or “very much improved”. Treatment success was achieved by 83.3% (n = 10) of patients at week 16 (primary end‐point) with CGI evaluated as “very much improved” (n = 9) and “much improved” (n = 1). Moreover, the area of erythema with pustules improved as early as week 1 and resolved by week 16 in most of the patients. The improvements were sustained throughout 52 weeks. Over the 52‐week treatment period, secukinumab was well tolerated with no unexpected safety signals. Nasopharyngitis, urticaria, diabetes mellitus and arthralgia were the frequent adverse events reported. The data from this study shows that secukinumab can become one of the potent treatment options for GPP.

Lamellar Granule Secretion Starts before the Establishment of Tight Junction Barrier for Paracellular Tracers in Mammalian Epidermis

Defects in epidermal barrier function and/or vesicular transport underlie severe skin diseases including ichthyosis and atopic dermatitis. Tight junctions (TJs) form a single layered network in simple epithelia. TJs are important for both barrier functions and vesicular transport. Epidermis is stratified epithelia and lamellar granules (LGs) are secreted from the stratum granulosum (SG) in a sequential manner. Previously, continuous TJs and paracellular permeability barriers were found in the second layer (SG2) of SG in mice, but their fate and correlation with LG secretion have been poorly understood. We studied epidermal TJ-related structures in humans and in mice and found occludin/ZO-1 immunoreactive multilayered networks spanning the first layer of SG (SG1) and SG2. Paracellular penetration tracer passed through some TJs in SG2, but not in SG1. LG secretion into the paracellular tracer positive spaces started below the level of TJs of SG1. Our study suggests that LG-secretion starts before the establishment of TJ barrier in the mammalian epidermis.

In Vitro and In Vivo Transfer of bcl-2 Gene into Keratinocytes Suppresses UVB-induced Apoptosis¶

Bcl‐2 is a member of the large Bcl‐2 family and protects cells from apoptosis. Ultraviolet B (UVB) irradiation induces apoptosis of keratinocytes that is known as “sunburn cells.” Previously we reported that UVB irradiation induces apoptosis accompanied by sequential activation of caspase 8, 3 and 1 in keratinocytes, and that the process is inhibited by various caspase inhibitors. Using bcl‐2–expressing adenovirus vector we investigated the effect of Bcl‐2 on UVB‐induced apoptosis. Adenovirus vector efficiently introduced bcl‐2 gene in cultured normal mouse keratinocytes (NMK cells); almost all NMK cells (1 × 106) were transfected at 1 × 108 plaque‐forming unit (PFU)/mL. Bcl‐2–transfected NMK cells were significantly resistant to UVB‐induced apoptosis with the suppressive effect dependent on the Bcl‐2 expression level. Following UVB irradiation caspase 8, 3 and 9 activities were stimulated in NMK cells, whereas in bcl‐2–transfected cells only caspase 8, but not caspase 3 or 9, activity was stimulated. In order to investigate the effect of Bcl‐2 in vivo topical application of Ad‐bcl‐2 on tape‐stripped mouse skin was performed. Following the application Bcl‐2 was efficiently overexpressed in almost all viable keratinocytes. The expression was transient with the maximal expression of Bcl‐2 on the first day following the application of 1 × 109 PFU in 200 μL. The introduced Bcl‐2 remained at least for 6 days. UVB irradiation (1250 J/m2) induced apoptosis within 12 h and the maximal effect was observed at 24 h in control mouse skin. Both bcl‐2–transfected and topical caspase 3 inhibitor‐treated mice skin were resistant to UVB‐induced apoptosis. The suppressive effect of Bcl‐2 was more potent than that of caspase 3 inhibitor application. Topical application of empty adenovirus vector alone had no effect on Bcl‐2 expression or UVB‐induced apoptosis. These results indicate that adenovirus vector is an efficient gene delivery system into keratinocytes and that Bcl‐2 is a potent inhibitor of UVB‐induced apoptosis both in vitro and in vivo.

Increased expression of mRNAs for microtubule disassembly molecules during nerve regeneration.

The mRNA expression of the microtubule disassembly molecules (SCG10, stathmin, SCLIP and RB3) in response to nerve injury was examined using a rat hypoglossal nerve injury model. After nerve injury prominent increase in mRNA expression of SCG10, stathmin and RB3 was observed, while only slight increase in SCLIP mRNA was observed in injured motor neurons. The increase in SCG10 and RB3 mRNA expression was quicker than that of stathmin and SCLIP. All the elevated signals decreased gradually to control levels by 4 weeks after nerve injury.

Developmental alteration of nerve injury induced glial cell line-derived neurotrophic factor (GDNF) receptor expression is crucial for the determination of injured motoneuron fate

Axotomy‐induced neuronal death occurs in neonatal motoneurons, but not in adult rat. Here we demonstrated that during the course of postnatal development, nerve injury induced down‐regulation of the glial cell line‐derived neurotrophic factor (GDNF) receptor GFRα1 in axotomized hypoglossal motoneurons of rat are gradually converted to the adult up‐regulation pattern of response. The compensatory expression of GFRα1 specifically in the injured motoneurons of neonates by adenovirus succeeded in rescuing the injured neurons without an application of growth factors. To the contrary, the nuclear antisense RNA for GFRα1 expression accelerates the axotomy‐induced neuronal death in pups. These findings suggest that the receptor expression response after nerve injury is critical for the determination of injured motoneuron fate.

Inhibition of Ras extracellular-signal-regulated kinase (ERK) mediated signaling promotes ciliary neurotrophic factor (CNTF) expression in Schwann cells

Ciliary neurotrophic factor (CNTF) can prevent injury‐induced motor neuron death. However, it is also evident that expression of CNTF in Schwann cells is suppressed during nerve regeneration. In this report, we have addressed the mechanism underlying the down‐regulation of CNTF expression in injured nerves using a mouse Schwann cell line IMS32 and mouse sciatic nerve. In IMS32 cells, activation of the Ras extracellular‐signal‐regulated kinase (ERK) pathway by adenoviral vector‐mediated expression of dominant active MEK1 did not alter a basal level of CNTF expression, whereas inhibition of the Ras‐ERK pathway by using adenoviral vectors resulted in a marked increase in CNTF expression. This inverse relation between before and after axotomy was also observed in mouse sciatic nerve. In the axotomized sciatic nerve, the phosphorylated ERK was markedly increased; in contrast, the expression of CNTF was markedly decreased. These findings suggest that an inactive state of ERK is crucial for the CNTF expression in Schwann cells, and that activation of ERK following nerve injury critically influences the expression of CNTF. This might well explain why CNTF is highly expressed in quiescent Schwann cells in the peripheral nervous system, and also why CNTF is not abundant in axotomized nerves or cultured Schwann cells in which the proliferation signal is obviously active.

Increased plasma resistin and decreased omentin levels in Japanese patients with psoriasis

Psoriasis is associated with obesity accompanied by insulin resistance. A recent study disclosed increased plasma resistin and decreased plasma omentin levels in obesity. Few studies of plasma levels of resistin and omentin are available in psoriasis. We analyzed plasma levels of resistin and omentin in psoriasis and compared them with those of healthy controls. Evaluation of plasma levels of resistin and omentin was performed by enzyme-linked immunosorbent assay (ELISA) for 62 psoriasis patients and 58 healthy controls. The severity of psoriasis was evaluated by psoriasis area and severity index (PASI) score. Plasma levels of resistin were significantly increased in psoriasis as compared with those of healthy controls. In contrast, plasma levels of omentin were significantly decreased in psoriasis patients. Plasma levels of resistin and omentin were positively and negatively correlated with PASI scores, respectively. After the treatment of psoriasis, resistin levels were decreased and omentin levels were increased, respectively, compared with those of pretreated. Plasma levels of resistin and omentin might be useful for evaluating the disease activity of psoriasis.