Epidermal phospholipase Cδ1 regulates granulocyte counts and systemic interleukin-17 levels in mice.

Phospholipase C is a key enzyme in phosphoinositide turnover. Although its functions have been extensively studied at the cellular level, many questions remain concerning its functions at the organ and individual animal levels. Here we demonstrate that mice lacking phospholipase Cδ1 develop granulocytosis associated with elevated serum levels of the granulopoietic cytokine interleukin-17. Re-introduction of phospholipase Cδ1 into keratinocytes of phospholipase Cδ1-deficient mice reverses this phenotype, whereas conditional ablation of phospholipase Cδ1 in keratinocytes recreates it. Interleukin-17 and its key upstream regulator interleukin-23 are also upregulated in epidermis. Loss of phospholipase Cδ1 from keratinocytes causes features of interleukin-17-associated inflammatory skin diseases. Phospholipase Cδ1 protein is downregulated in the epidermis of human psoriatic skin and in a mouse model of psoriasis. These results demonstrate that phosphoinositide turnover in keratinocytes regulates not only local inflammatory responses but also serum cytokine levels and systemic leukocyte counts, and affects distant haematopoietic organs.

Phospholipase C-delta1 is an essential molecule downstream of Foxn1, the gene responsible for the nude mutation, in normal hair development.

Nude mice exhibit athymia and hairlessness by a loss-of-function mutation in the transcription factor Foxn1 gene. Although the immunological functions of Foxn1 have been studied intensively, there have been relatively few studies of its functions in skin. Foxn1 regulates expression of hair keratins, which is essential for normal hair structure; however, how Foxn1 regulates hair keratin expression and hair formation is largely unknown. In the present study, we found that mice lacking phospholipase C (PLC)-delta1, a key molecule in the phosphoinositide signaling pathway, and nude mice show similar hair abnormalities, such as lack of cuticle and bending. We also found that expression of hair keratins was remarkably decreased in skin of PLC-delta1 knockout mice. Furthermore, expression of PLC-delta1 was induced in Foxn1-transfected U2OS cells. In addition, we showed that PLC-delta1 expression was remarkably decreased in skin of nude mice. In skin and keratinocytes of nude mice as well as PLC-delta1 KO mice, activation of PLC downstream effectors, such as PKC and nuclear factor of activated T cells, was impaired. These results indicate that PLC-delta1 is an essential molecule downstream of Foxn1 in normal hair formation, and strongly suggest that hairlessness in nude mice is caused by insufficient expression of PLC-delta1.

Lack of phospholipase C-delta1 induces skin inflammation.

Phospholipase C (PLC) is a key enzyme in phosphoinositide signaling. We previously generated PLC-delta1 knockout (KO) mice and found that these mice showed remarkable hair loss caused by abnormalities in hair follicle structures. Here we show that the skin of PLC-delta1 KO mice displays typical inflammatory phenotypes, including increased dermal cellularity, leukocyte infiltration, and expression of pro-inflammatory cytokines. In addition, exogenously expressed PLC-delta1 attenuates lipopolysaccharide-induced expression of IL-1beta, a pro-inflammatory cytokine, in an enzymatic activity-dependent manner. Furthermore, suppression of skin inflammation by anti-inflammatory reagents cured the epidermal hyperplasia in PLC-delta1 KO mice. Taken together, these results indicate that lack of PLC-delta1 induces skin inflammation and that the epidermal hyperplasia in PLC-delta1 KO mice is caused by skin inflammation. Our results also suggest that PLC-delta1 regulates homeostasis of the immune system in skin.

Analysis of 95 cases of squamous cell carcinoma of the external and middle ear.

OBJECTIVE: To analyze the clinical characteristics, 5-year survival, and prognostic factors of squamous cell carcinoma (SCC) of the external and middle ear. METHODS: A multi institutional study. Ninety five cases of patients from 10 institutions were reviewed on their age and sex distribution, initial complaints, stages, tumor locations, treatments, and outcomes. Prognostic factors were discussed based on the Pittsburgh staging system. RESULTS: This disease seems to appear in the elderly with a peak age of 50-69 years. Males appear to be more predisposed than females with an odd ratio of 1.7. The initial complaints were not typical, while 12.6% of patients presented a history of recurrent otitis externa or chronic otitis media. Regional metastasis was recognized in 13.7% of patients, while no distant metastasis was confirmed. SCC located in the external ear could be detected in an earlier stage than that in the middle ear. The overall 5-year survival was 66.8% in total, and decreased significantly with stage. SCC in stages I and II was susceptible to each therapeutic strategy with a 5-year survival of 100%. Operation combined with radiotherapy and/or chemotherapy was the major treatment for stages III and IV SCC, while radiotherapy and chemotherapy were applied mainly for those who had been considered inappropriate for operation. The overall survival was 67.2% for stage III and 29.5% for stage IV, and operation with pathologically tumor free margin could improve the survival to 72.7% when combined with radio- and chemotherapy. Stage, completeness of operation with tumor free margin, recurrence, and metastasis have significant influence on survival. CONCLUSION: Local infiltration seems to be the main behavior of SCC in the external and middle ear. Early diagnosis and treatment were important because SCC in the earlier stage is susceptible to be cured. For tumors of advanced stage, operation should be performed with pathologically tumor free margin, and operation combined with radiotherapy and chemotherapy could improve the survival. Tumor stage adds more influence on survival than its location. Recurrence and metastasis mainly occur in advanced stages and result in a poor survival.

Phospholipase Cdelta4 associates with glutamate receptor interacting protein 1 in testis.

We reported previously that phospholipase C (PLC) delta4 is required for calcium mobilization in the zona pellucida-induced acrosome reaction in sperm. Here we focused on the function of the C2 domain of PLCdelta4 and report that glutamate receptor-interacting protein1 (GRIP1) was identified as a binding protein of the PLCdelta4-C2 domain on yeast two-hybrid screening. Physiological interaction of GRIP1 with PLCdelta4 in mouse testis was confirmed by immunoprecipitation with anti-PLCdelta4 antibodies and the association seemed to correlate with the maturation stage of sperm. We also determined that a PDZ-binding motif at the C-terminus of the PLCdelta4-C2 domain is responsible for GRIP1 binding, whereas the sixth or seventh PDZ domain of GRIP1 is essential and sufficient for association with the PLCdelta4-C2 domain. These results indicate that PLCdelta4 binds via its C2 domain to the PDZ6 or PDZ7 domain of GRIP1, and that this association may play a role in spermatogenesis.

The prevalence of obesity and its relationship with lifestyle factors in Jamaica.

We surveyed the prevalence of obesity in the general population in Jamaica, and examined the relationship between it and lifestyle. The survey population consisted of 1,935 inhabitants in Jamaica, whose body weight, height, marital status, educational history, employment status and other obesity-associated lifestyle factors were surveyed. Six major findings emerged. The first finding is that the proportion of obesity in women was very high, and there was a big gender difference. Secondly, a lower prevalence towards obesity was associated with cohabitation of the subjects in both genders, and higher educational levels in female subjects. Thirdly, the proportion of the subjects who considered their weight to be quite acceptable was higher in the obese/overweight groups in both genders. Fourthly, exercise frequency showed a negative correlation with the body mass index (BMI) in men, and the frequency of exercising was apparently lower in women than in men. Fifthly, as for dietary factors, in both genders vegetables showed a negative correlation with the BMI. Sixthly, non-smokers were also associated with a lower obesity prevalence in men. In conclusions, these findings suggest that social and lifestyle factors such as the educational level, marital status and dietary habits of the general population influence Jamaican obesity.

Lecithinized brain-derived neurotrophic factor promotes the differentiation of embryonic stem cells in vitro and in vivo.

The addition of lecithin molecules to brain-derived neurotrophic factor (BDNF) has been reported to markedly enhance its pharmacological effect in vivo. In the current study, we show that lecithinized BDNF (PC-BDNF) has a higher affinity than BDNF for neural precursor cells. Although BDNF only slightly increased the expression of the genes for Mash-1, p35, 68 kDa neurofilament, and TrkB receptor, PC-BDNF caused a significant increase in their expression. PC-BDNF also increased the level of neurofilament protein and dramatically increased TrkB mRNA gene expression, which was followed by a sustained activation of the p42/p44 extracellular-regulated kinases. Finally, transplantation of PC-BDNF-treated cells was more effective than BDNF-treated cells at improving impaired motor function caused by spinal cord injury. These findings showed that PC-BDNF has a better potential than BDNF for promoting neural differentiation, partly due to a higher cellular affinity. Furthermore, PC-BDNF-treated cells could be useful for transplantation therapy for central nervous system injuries.

Brain-derived neurotrophic factor bound with lecithin derivative showed a markedly enhanced pharmacological potency due to its potent cell membrane affinity followed by prolonged MAPK activation.

We synthesized lecithinized brain-derived neurotrophic factor (lecithinized-BDNF), in which an average of three molecules of a lecithin derivative were bound to recombinant human BDNF. We evaluated its pharmacological activity in C57BL/KsJ-db/db mice, and assessed its targetability and affinity for the nervous system. Subcutaneously administered lecithinized-BDNF markedly reduced the plasma glucose level, food intake, and body weight in C57BL/KsJ-db/db diabetic mice. Its potency was more than 20 times greater than that of unmodified BDNF. We then studied the mechanism for the markedly enhanced pharmacological activity. In vitro cell growth activity of lecithinized-BDNF using the MTT assay was lower than unmodified BDNF, probably due to steric hindrance of the lecithin moieties. While the plasma BDNF level after subcutaneous administration of lecithinized-BDNF was not higher compared with unmodified BDNF. However, higher amount of lecithinized-BDNF accumulated in the spinal cord was observed. Lastly, we found that in vitro binding capacity of lecithinized-BDNF for PC-pAB1 neural cells was much higher than unmodified BDNF. Moreover, lecithinized-BDNF bound to PC-pAB1 cells did not exchange with an excessive amount of unmodified BDNF or an excess of lecithinized-BDNF. PC-pAB1 cells treated with lecithinized-BDNF showed sustained mitogen-activated protein kinase (MAPK, ERK1/2) activation. These data would indicate that the high affinity of lecithinized-BDNF for the target cells, followed by prolonged MAPK activation, would play an important role in its potent pharmacological activity.