A novel semi-dominant mutation in brassinosteroid signaling kinase1 increases stomatal density

Stomata play a pivotal role in balancing CO2 uptake for photosynthesis and water loss via transpiration. Thus, appropriate regulation of stomatal movement and its formation are crucial for plant growth and survival. Red and blue light induce phosphorylation of the C-terminal residue of the plasma membrane (PM) H+-ATPase, threonine, in guard cells, generating the driving force for stomatal opening. While significant progress has been made in understanding the regulatory mechanism of PM H+-ATPase in guard cells, the regulatory components for the phosphorylation of PM H+-ATPase have not been fully elucidated. Recently, we established a new immunohistochemical technique for detecting guard-cell PM H+-ATPase phosphorylation using leaves, which was expected to facilitate investigations with a single leaf. In this study, we applied the technique to genetic screening experiment to explore novel regulators for the phosphorylation of PM H+-ATPase in guard cells, as well as stomatal development. We successfully performed phenotyping using a single leaf. During the experiment, we identified a mutant exhibiting high stomatal density, jozetsu (jzt), named after a Japanese word meaning ‘talkative’. We found that a novel semi-dominant mutation in BRASSINOSTEROID SIGNALING KINASE1 (BSK1) is responsible for the phenotype in jzt mutant. The present results demonstrate that the new immunohistochemical technique has a wide range of applications, and the novel mutation would provide genetic tool to expand our understanding of plant development mediated by brassinosteroid signaling

Inhibition of light-induced stomatal opening by allyl isothiocyanate does not require guard cell cytosolic Ca2+ signaling

The glucosinolate-myrosinase system is a well-known defense system that has been shown to induce stomatal closure in Brassicales. Isothiocyanates are highly reactive hydrolysates of glucosinolates, and an isothiocyanate, allyl isothiocyanate (AITC), induces stomatal closure accompanied by elevation of free cytosolic Ca2+ concentration ([Ca2+](cyt)) in Arabidopsis. It remains unknown whether AITC inhibits light-induced stomatal opening. This study investigated the role of Ca2+ in AITC-induced stomatal closure and inhibition of light-induced stomatal opening. AITC induced stomatal closure and inhibited light-induced stomatal opening in a dose-dependent manner. A Ca2+ channel inhibitor, La3+, a Ca(2+)chelator, EGTA, and an inhibitor of Ca2+ release from internal stores, nicotinamide, inhibited AITC-induced [Ca2+](cyt) elevation and stomatal closure, but did not affect inhibition of light-induced stomatal opening. AITC activated non-selective Ca2+-permeable cation channels and inhibited inward-rectifying K+ (K-in(+)) channels in a Ca2+-independent manner. AITC also inhibited stomatal opening induced by fusicoccin, a plasma membrane H+-ATPase activator, but had no significant effect on fusicoccin-induced phosphorylation of the penultimate threonine of H+-ATPase. Taken together, these results suggest that AITC induces Ca2+ influx and Ca2+ release to elevate [Ca2+](cyt), which is essential for AITC-induced stomatal closure but not for inhibition of K-in(+) channels and light-induced stomatal opening

The Rax homeoprotein in Müller glial cells is required for homeostasis maintenance of the postnatal mouse retina

Müller glial cells, which are the most predominant glial subtype in the retina, play multiple important roles, including the maintenance of structural integrity, homeostasis, and physiological functions of the retina. We have previously found that the Rax homeoprotein is expressed in postnatal and mature Müller glial cells in the mouse retina. However, the function of Rax in postnatal and mature Müller glial cells remains to be elucidated. In the current study, we first investigated Rax function in retinal development using retroviral lineage analysis and found that Rax controls the specification of late-born retinal cell types, including Müller glial cells in the postnatal retina. We next generated Rax tamoxifen–induced conditional KO (Rax iCKO) mice, where Rax can be depleted in mTFP-labeled Müller glial cells upon tamoxifen treatment, by crossing Raxflox/flox mice with Rlbp1-CreERT2 mice, which we have produced. Immunohistochemical analysis showed a characteristic of reactive gliosis and enhanced gliosis of Müller glial cells in Rax iCKO retinas under normal and stress conditions, respectively. We performed RNA-seq analysis on mTFP-positive cells purified from the Rax iCKO retina and found significantly reduced expression of suppressor of cytokine signaling-3 (Socs3). Reporter gene assays showed that Rax directly transactivates the Socs3 promoter. We observed decreased expression of Socs3 in Müller glial cells of Rax iCKO retinas by immunostaining. Taken together, the present results suggest that Rax suppresses inflammation in Müller glial cells by transactivating Socs3. This study sheds light on the transcriptional regulatory mechanisms underlying retinal Müller glial cell homeostasis.Yoshimoto T., Chaya T., Varner L.R., et al. The Rax homeoprotein in Müller glial cells is required for homeostasis maintenance of the postnatal mouse retina. Journal of Biological Chemistry 299, 105461 (2023); https://doi.org/10.1016/j.jbc.2023.105461

A Kinome-Wide Synthetic Lethal CRISPR/Cas9 Screen Reveals That mTOR Inhibition Prevents Adaptive Resistance to CDK4/CDK6 Blockade in HNSCC.

UNLABELLED: The comprehensive genomic analysis of the head and neck squamous cell carcinoma (HNSCC) oncogenome revealed the frequent loss of p16INK4A (CDKN2A) and amplification of cyclin D1 genes in most human papillomavirus-negative HNSCC lesions. However, cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors have shown modest effects in the clinic. The aberrant activation of the PI3K/mTOR pathway is highly prevalent in HNSCC, and recent clinical trials have shown promising clinical efficacy of mTOR inhibitors (mTORi) in the neoadjuvant and adjuvant settings but not in patients with advanced HNSCC. By implementing a kinome-wide CRISPR/Cas9 screen, we identified cell-cycle inhibition as a synthetic lethal target for mTORis. A combination of mTORi and palbociclib, a CDK4/6-specific inhibitor, showed strong synergism in HNSCC-derived cells in vitro and in vivo. Remarkably, we found that an adaptive increase in cyclin E1 (CCNE1) expression upon palbociclib treatment underlies the rapid acquired resistance to this CDK4/6 inhibitor. Mechanistically, mTORi inhibits the formation of eIF4G-CCNE1 mRNA complexes, with the consequent reduction in mRNA translation and CCNE1 protein expression. Our findings suggest that mTORi reverts the adaptive resistance to palbociclib. This provides a multimodal therapeutic option for HNSCC by cotargeting mTOR and CDK4/6, which in turn may halt the emergence of palbociclib resistance. SIGNIFICANCE: A kinome-wide CRISPR/Cas9 screen identified cell-cycle inhibition as a synthetic lethal target of mTORis. A combination of mTORi and palbociclib, a CDK4/6-specific inhibitor, showed strong synergistic effects in HNSCC. Mechanistically, mTORis inhibited palbociclib-induced increase in CCNE1

Periostin Promotes Tumor Lymphangiogenesis

Background: Metastasis to regional lymph nodes via lymphatic vessels plays a key role in cancer progression. Tumor lymphangiogenesis is known to promote lymphatic metastasis, and vascular endothelial growth factor C (VEGF-C) is a critical activator of tumor lymphangiogenesis during the process of metastasis. We previously identified periostin as an invasion- and angiogenesis-promoting factor in head and neck squamous cell carcinoma (HNSCC). In this study, we discovered a novel role for periostin in tumor lymphangiogenesis. Methods and Findings: Periostin overexpression upregulated VEGF-C mRNA expression in HNSCC cells. By using conditioned media from periostin-overexpressing HNSCC cells, we examined tube formation of lymphatic endothelial cells. Conditioned media from periostin-overexpressing cells promoted tube formation. To know the correlation between periostin and VEGF-C, we compared Periostin expression with VEGF-C expression in 54 HNSCC cases by immunohistochemistry. Periostin expression was correlated well with VEGF-C expression in HNSCC cases. Moreover, correlation between periostin and VEGF-C secretion was observed in serum from HNSCC patients. Interestingly, periostin itself promoted tube formation of lymphatic endothelial cells independently of VEGF-C. Periostin-promoted lymphangiogenesis was mediated by Src and Akt activity. Indeed possible correlation between periostin and lymphatic status in periostin-overexpressing xenograft tumors and HNSCC cases was observed. Conclusions: Our findings suggest that periostin itself as well as periostin-induced upregulation of VEGF-C may promote lymphangiogenesis. We suggest that periostin may be a marker for prediction of malignant behaviors in HNSCC and a potential target for future therapeutic intervention to obstruct tumoral lymphatic invasion and lymphangiogenesis in HNSCC patients

Ameloblastin induces tumor suppressive phenotype and enhances chemosensitivity to doxorubicin via Src-Stat3 inactivation in osteosarcoma

Ameloblastin (AMBN), the most abundant non-amelogenin enamel matrix protein, plays a role in ameloblast differentiation. Previously, we found that AMBN promoted osteogenic differentiation via the interaction between CD63 and integrin β1, leading to the inactivation of Src; however, how AMBN affects the malignant behavior of osteosarcoma is still unclear. Osteosarcoma affects the bone and is associated with poor prognosis because of the high rate of pulmonary metastases and drug resistance. Here we demonstrated that stable overexpression of AMBN induced apoptosis and suppressed colony formation and cell migration via the inactivation of Src-Stat3 pathway in human osteosarcoma cells. Moreover, AMBN induced chemosensitivity to doxorubicin. Thus, AMBN induced a tumor suppressive phenotype and chemosensitivity to doxorubicin via the AMBN-Src-Stat3 axis in osteosarcoma. Indeed, immunohistochemical expression of AMBN was significantly correlated with better outcome of osteosarcoma patients. Our findings suggest that AMBN can be a new prognostic marker and therapeutic target for osteosarcoma combined with conventional doxorubicin treatment

有機酸で処理したリン鉱石中のリンに対するイタリアンライグラスの反応

スリランカとトーゴー産のリン鉱石に5Mのクエン酸、シュウ酸、酒石酸溶液を1:2の割合で加え、3日間反応させた。シュウ酸と酒石酸で処理したリン鉱石は容易に乾燥し、ほとんど全てのリン酸が水溶性となった。クエン酸で処理したリン鉱石は乾燥しにくく、また水溶性リン酸の割合も全リン酸の50%程度であった。これら有機酸で処理したリン鉱石のリン源としての有効性を、イタリアンライグラスを供試作物とし、マサ土(花崗岩風化土壌)を用いた土耕ポット試験で過燐酸石灰と比較した。クエン酸処理したスリランカ産リン鉱石を除き、他の全ての有磯酸処理リン鉱石は播種後168日目のイタリアンライグラスの乾物収量を過燐酸石灰と同程度に増加させた。酒石酸で処理したトーゴー産リン鉱石におけるリンの回収率は58%で、過燐酸石灰(52%)や酒石酸処理スリランカ産リン鉱石(53%)より有意に高かった。有機酸処理したリン鉱石を与えた区の跡地土壌の可給態リン(Olsen P)濃度とpHは、過燐酸石灰施与区や無リン酸区(対照区)より有意に高かった。実験期間を通じてイタリアンライグラスの生育と養分吸収に及ぼす有機酸処理リン鉱石施与の悪影響は認められなかった。以上の結果から、酸性土壌に生育する作物に対し、少なくとも酒石酸処理したリン鉱石のリン源として有効性は過燐酸石灰より良好であると判断された。Two phosphate rocks (PRs) from Sri Lanka and Togo were acidulated with 5 M of citric, oxalic or tartaric acid at 2:1 acid:rock ratio for three days. The resulting materials treated with oxalic and tartaric acid were easily dried up and nearly all the P was in water soluble form. The PRs treated with citric acid were rather difficult to dry and contained about 50% of the total P in water soluble form. The effectiveness of the materials as phosphorus sources were compared with that of single superphosphate in acidic granitic regosol with Italian ryegrass as the test crop. All the acid treatments were as effective as single superphosphate in increasing the dry matter yield of Italian ryegrass at 168 days after planting except the citric-acid treated Sri Lanka PR. Phosphorus recovery by the plants from tartaric-acid treated Togo PR was 58%, which was significantly greater (P<0.05) than those from single superphosphate (52%) and tartaric-acid treated Sri Lanka PR (53%). Significantly higher levels of available P (OLSEN P) and pH were found in the cropped soils which received the organic-acid treated PRs than those of the soils which received single superphosphate and control at the end of the experiment. No adverse effect of the organic-acid treated PRs on the growth and nutrient uptake by Italian ryegrass was observed throughout the growth period. These results indicate that at least the tartaric-acid treated PRs could be better sources of P to crops growing on acid soils than single superphosphate