Effect of Day Length, Supplemental Lighting Strength, Shading Period and Minimum Night Temperature on Occurrence of Abnormal Inflorescence in Gypsophila paniculata ‘Altair’

As occurrence of abnormal inflorescence in Gypsophila paniculata ‘Altair’ is caused by environmental conditions, effects of day length, supplemental lighting strength, shading period and minimum night temperature on occurrence of abnormal inflorescence were investigated. Abnormal inflorescence was classified into four types : normal, pattern 1 (Short-flower stalk), pattern 2 (Coalescent two-flower stalk) and pattern 3 (Looping and irregular-flower stalk). Neither of 12h, 16h, 20h or 24h day length by fluorescent lamp, nor 24h by incandescent lamp affected occurrence of abnormal inflorescence. Effects of four levels of light intensity (fluorescent lamp : PPFD 1μmol・m−2・s−1, incandescent lamp : PPFD 3μmol・m−2・s−1, metal halide lamp : PPFD 14μmol・m−2・s−1 and high-pressure sodium lamp : PPFD 48μmol・m−2・s−1) were examined in 16h photoperiod. Occurrence of abnormal inflorescence was not affected by different light intensities, neither was it affected by shading period. Occurrence of abnormal inflorescence at 15°C was however significantly reduced compared to that at 8°C. In particular, patterns 2 and 3 at 15°C were significantly reduced compared to those at 8°C. There was a strong negative correlation between average night temperature from starting the treatment to flower budding (7.1°C, 9.0°C, 9.2°C, 11.6°C and 16.4°C) and incidence of pattern 3 (13.1%, 8.7%, 7.1%, 1.1% and 0.7%). Therefore, as average night temperature increased, occurrence of abnormal inflorescence decreased. The results show that low night temperature may be the main factor inducing occurrence of abnormal inflorescence.シュッコンカスミソウ‘アルタイル’の形態異常花序の発生には環境要因が関与していると考えられたので，日長，補光強度，遮光時期および最低夜温が形態異常花序発生に及ぼす影響を調査した．形態異常程度は４種類のパターン (０：正常，１：茎が短いもの，２：２本の茎が癒着，3：ひどく湾曲し変形したもの) に分類し，その影響を受けた小花の割合を求めた．蛍光灯による日長処理（12時間，16時間，20時間，24時間）や白熱灯による日長処理（自然日長，24時間）は形態異常花序発生率に影響を及ぼさなかった．蛍光灯（PPFD 1μmol・m-2・s-1），白熱灯（PPFD 3μmol・m-2・s-1），メタルハライドランプ（PPFD 14μmol・m-2・s-1），高圧ナトリウムランプ（PPFD 48μmol・m-2・s-1）を用いて16時間の補光を行った．異なる光源による光強度でも形態異常発生率に一定の傾向は認められなかった．遮光時期を変えても形態異常発生率に一定の傾向は認められなかった． 最低夜温を15℃に上げると８℃区と比較して15℃区の形態異常発生は大きく減少した．特にパターン２と３の発生率は大幅に低下した．各実験の処理開始から発蕾までの平均夜温（7.1℃，9.0℃，9.2℃，11.6℃，16.4℃）と，パターン３の形態異常発生率（13.1%，8.7%，7.1%，1.1%，0.7%）との間に高い負の相関（R2＝0.849）が認められ，処理開始から発蕾までの平均夜温が高いほど形態異常発生率は低下した．以上のことから，形態異常花序発生には夜間の温度が大きく関与しているのではないかと推察された

Serum affects keratinization and tight junctions in three-dimensional cultures of the mouse keratinocyte cell line COCA through retinoic acid receptor-mediated signaling.

Vitamin A, which is found in serum, is known to affect keratinocyte proliferation, epidermal differentiation, and keratinization. In mice, stratified epithelia in the oral cavity, esophagus, and forestomach are keratinized; however, these epithelia are not keratinized in humans. Several studies have reported that three-dimensional (3D) cultures of human keratinocytes in serum-containing medium could form keratinized epithelia. Here, we evaluated the effects of serum on the morphology, expression, and localization of differentiation markers and tight junction proteins, and paracellular permeability in 3D cultures of mouse keratinocytes. We found that only 0.1% calcium-depleted serum inhibited keratinization and induced a change in the expression of differentiation marker proteins from loricrin to keratin 4; the inhibition of retinoic acid receptor-mediated signaling reversed these changes. Furthermore, the serum reduced claudin-1 protein expression and prevented its localization at occludin-positive spots on the surface of 3D cultures. On the other hand, the serum increased the protein expression of claudin-4, occludin, zonula occludens-1, and E-cadherin. These changes may contribute to the reduction of the transepithelial electrical resistance by approximately half. In conclusion, mouse keratinocytes derived from the epidermis formed non-keratinized structures in 3D cultures in response to vitamin A in serum. The results suggest that retinoic acid receptor-mediated signaling may be inhibited in the mouse epithelia in the oral cavity, esophagus, and forestomach as well as the epidermis, leading to the keratinization of these epithelia.福岡歯科大学2018年

Anisomycin, a JNK and p38 activator, suppresses cell-cell junction formation in 2D cultures of K38 mouse keratinocyte cells and reduces claudin-7 expression, with an increase of paracellular permeability in 3D cultures.

Keratinocytes in the oral mucosal epithelium, which is a non-keratinized stratified epithelium, are exposed to various stimuli from the oral cavity. JNK and p38 are stress-activated mitogen-activated protein kinases (MAPKs) that are phosphorylated by various stimuli and are involved in the assembly and disassembly of tight junctions (TJs) in keratinocytes. Therefore, we investigated the effects of stress-activated MAPKs on TJs in a mouse keratinocyte cell line during cell-cell junction formation in two-dimensional (2D) cultures or stratification to form non-keratinized epithelium in 3D cultures. In 2D cultures, calcium induced zipper-like staining for ZO-1 at 2 h and string-like staining for ZO-1 at 12 h, which indicated immature and mature cell-cell junctions, respectively. Anisomycin (AM), a JNK and p38 activator, inhibited formation of string-like staining for ZO-1, whereas inhibition of JNK, but not p38, after AM treatment restored string-like staining for ZO-1, although claudins (CLDNs) 4, 6, and 7 did not completely colocalize to ZO-1-positive sites. In 3D cultures, AM treatment for 2 weeks activated only p38, suppressed flattening of the superficial cells, removed CLDN7 from ZO-1-positive spots on the surface of 3D cultures, which represent TJs, and decreased transepithelial electrical resistance. Thus, short-term AM treatment inhibited maturation of cell-cell junctions by JNK, but not p38, activation. p38 activation by long-term AM treatment affected morphology of stratified structures and paracellular permeability, which was increased by CLDN7 removal from TJs. Various chronic stimuli that activate stress-activated MAPKs may weaken the keratinocyte barrier and be involved in TJ-related diseases.福岡歯科大学2018年

The reduced susceptibility of mouse keratinocytes to retinoic acid may be involved in the keratinization of oral and esophageal mucosal epithelium.

Keratinocytes take up serum-derived retinol (vitamin A) and metabolize it to all-trans-retinoic acid (atRA), which binds to the nuclear retinoic acid receptor (RAR). We previously reported that serum-affected keratinocyte differentiation and function; namely, it inhibited keratinization, decreased loricrin (LOR) and claudin (CLDN) 1 expression, increased keratin (K) 4 and CLDN4 levels, and reduced paracellular permeability in three-dimensional (3D) cultures of mouse keratinocytes (COCA). Contrarily, RAR inhibition reversed these changes. Here, we aimed to examine whether atRA exerted the same effects as serum, and whether it was involved in the differential oral mucosa keratinization among animal species. Porcine oral mucosal keratinocytes, which form non-keratinized epithelium in vivo, established keratinized epithelium in 3D cultures. Both mouse and porcine sera induced non-keratinized epithelium at 0.1% in COCA 3D cultures. Although atRA caused the same changes as serum, its effective concentration differed. atRA inhibited keratinization at 0.1 nM and 1 nM in porcine or human keratinocytes and COCA, respectively. Furthermore, atRA upregulated CLDN7 in the cytoplasm but not in cell-cell contacts. These atRA-induced changes were reverted by RAR inhibition. The results indicate that serum-induced changes are probably due to the effect of serum-derived atRA, and that mouse keratinocytes require higher atRA concentrations to suppress keratinization than porcine and human keratinocytes. We propose that the lower susceptibility of mouse keratinocytes to atRA, rather than a lower retinol concentration, is a possible reason for the keratinization of mouse oral mucosal epithelium.福岡歯科大学2019年

Inhibition of retinoid X receptor improved the morphology, localization of desmosomal proteins and paracellular permeability in three-dimensional cultures of mouse keratinocytes.

Retinoic acid (RA) plays an important role in epithelial homeostasis and influences the morphology, proliferation, differentiation and permeability of epithelial cells. Mouse keratinocytes, K38, reconstituted non-keratinized stratified epithelium in three-dimensional (3D) cultures with serum, which contains retinol (a source of RA), but the morphology was different from in vivo epithelium. The formed epithelium was thick, with loosened cell-cell contacts. Here, we investigated whether the inhibition of RA receptor (RAR)/retinoid X receptor (RXR)-mediated signaling by an RXR antagonist, HX 531, improved K38 3D cultures in terms of morphology and intercellular junctions. The epithelium formed by 0.5 μM HX531 was thin, and the intercellular space was narrowed because of the restoration of the layer-specific distribution of desmoglein (DSG)-1, DSG3 and plakoglobin (PG). Moreover, the levels of desmosomal proteins and tight junction proteins, including DSG1, DSG2, DSG3, PG, claudin (CLDN)-1 and CLDN4 increased, but the adherens junction protein, E-cadherin, did not show any change. Furthermore, CLDN1 was recruited to occludin-positive cell-cell contacts in the superficial cells and transepithelial electrical resistance was increased. Therefore, K38 3D cultures treated with 0.5 μM HX531 provides a useful in vitro model to study intercellular junctions in the non-keratinized epithelium.福岡歯科大学2021年