Objective: To evaluate the function of Piezo1, an evolutionarily conserved mechanically activated channel, in periodontal ligament (PDL) tissue homeostasis under compressive loading. Materials and Methods: Primary human PDL cells (hPDLCs) were isolated, cultured, and then subjected to 2.0 g/cm 2 static compressive loading for 0.5, 3, 6, and 12 hours, respectively. The expressions of Piezo1 and osteoclastogenesis marker gene were assessed by semiquantitative reverse transcription-polymerase chain reaction. In addition, Piezo1 inhibitor, GsMTx4, was used to block the function of Piezo1, and tumor necrosis factor-a was also used as a positive control. After 12 hours of compressive loading the PDLCs were co-cultured with murine monocytic cell line RAW264.7. Immunofluorescence, western blot, enzyme-linked immunosorbent assay, and tartrate-resistant acid phosphatase staining were also used to test the potency of PDLCs to induce osteoclastogenesis and the activation of nuclear factor (NF)-kB. Results: Piezo1, cyclooxygenase-2, receptor activator of NF-kB ligand, and prostaglandin E2 were significantly upregulated under static compressive stimuli. GsMTx4 repressed osteoclastogenesis in the mechanical stress-pretreated PDLCs-RAW264.7 co-culture system. Furthermore, NF-kB signaling pathway was involved in the mechanical stress-induced osteoclastogenesis. Conclusions: Piezo1 exerts a transduction role in mechanical stress-induced osteoclastogenesis in hPDLCs. (Angle Orthod. 2015;85:87-94.)
Prolactin-releasing peptide (PrRP) and its structurally related peptide, Carassius Arg-Phe-amide peptide (C-RFa), have been reported to play similar roles in regulating food intake and pituitary functions in vertebrates. However, the identity, functionality, and expression of the receptor(s) for PrRP and C-RFa remain largely unknown in nonmammalian vertebrates, including birds. In this study, three receptors homologous to mammalian PrRP receptor (PrRPR), named cPrRPR1, cPrRPR2, and cC-RFaR, respectively, were cloned from chicken brain by RT-PCR. Using a pGL3-NFAT-RE-luciferase reporter system, we demonstrated that cPrRPR1 and cPrRPR2 expressed in Chinese hamster ovarian cells could be activated by cPrRP₂₀ and cC-RFa₂₀ potently, whereas cC-RFaR could only be activated effectively by cC-RFa₂₀ (EC₅₀, 0.11 nM), indicating that cPrRPR1 and cPrRPR2 can function as common receptors for PrRP and C-RFa, whereas cC-RFaR is a receptor specific to C-RFa. Using a pGL3-CRE-luciferase reporter system, cPrRPR1, cPrRPR2, and cC-RFaR expressed in Chinese hamster ovarian cells were also shown to activate intracellular protein kinase A signaling pathway upon cC-RFa₂₀ treatment (100 nM). Moreover, RT-PCR assay revealed that cPrRPR1, cPrRPR2, and cC-RFaR were widely expressed in most adult chicken tissues examined, including various regions of brain. These findings, together with evidence of PrRP and C-RFa encoded by separate genes in chicken, Xenopus, and zebrafish, and the differential expression of PrRP and C-RFa genes in chicken tissues, strongly suggest that PrRP and C-RFa may play similar yet distinctive roles in nonmammalian vertebrates, including chicken, and their actions are mediated by common receptor(s) or a specific C-RFa receptor.
The 2 structurally and functionally related peptides, neuropeptide B (NPB) and neuropeptide W (NPW), together with their receptor(s) (NPBWR1/NPBWR2) constitute the NPB/NPW system, which acts mainly on the central nervous system to regulate many physiological processes in mammals. However, little is known about this NPB/NPW system in nonmammalian vertebrates. In this study, the functionality and expression of this NPB/NPW system and its actions on the pituitary were investigated in chickens. The results showed that: 1) chicken NPB/NPW system comprises an NPB peptide of 28 amino acids (cNPB28), an NPW peptide of 23 or 30 amino acids (cNPW23/cNPW30), and their 2 receptors (cNPBWR1 and cNPBWR2), which are highly homologous to their human counterparts. 2) Using a pGL3-CRE-luciferase reporter system, we demonstrated that cNPBWR2 expressed in Chinese hamster ovary cells can be potently activated by cNPW23 (not cNPB28), and its activation inhibits the intracellular cAMP signaling pathway, whereas cNPBWR1 shows no response to peptide treatment, suggesting a crucial role of cNPBWR2 in mediating cNPW/cNPB actions. 3) Quantitative real-time PCR revealed that cNPW and cNPB are widely expressed in chicken tissues, including hypothalamus, whereas cNPBWR1 and cNPBWR2 are mainly expressed in brain or pituitary. 4) In accordance with abundant cNPBWR2 expression in pituitary, cNPW23 could dose dependently inhibit GH and prolactin secretion induced by GHRH and vasoactive intestinal polypeptide, respectively, in cultured chick pituitary cells, as monitored by Western blotting. Collectively, our data reveal a functional NPB/NPW system in birds and offer the first proof that NPW can act directly on pituitary to inhibit GH/prolactin secretion in vertebrates.
Objective: To test the hypothesis that young people's esthetic perception of dentition midline deviation or the threshold below which they find the deviation ''acceptable'' depends on the gender and face type of the person with the deviation and the gender of the evaluator. Materials and Methods: Facial images of six young subjects with three different face types were captured, and their dentition midlines were altered digitally. The images were evaluated by young people with no dental training. Statistical analysis was carried out to determine the threshold for acceptable dentition midline deviation and factors influencing perception. Results: The mean value for the threshold below which a deviation was judged ''acceptable'' was 2.403 mm (95% confidence interval, 2.315-2.491). The preferences of evaluators did not significantly depend on the direction of the deviation. Both male and female evaluators were significantly less tolerant of deviation in female subjects than in male subjects. However, female evaluators were significantly more tolerant of midline deviations in male subjects than were male evaluators. In addition, the same degree of deviation was most noticeable in male subjects with a tapered face type and least noticeable in female subjects with a square face type. Conclusions: The gender and face type of an individual with dentition midline deviation and the gender of the evaluator do affect young people's esthetic perception of a dentition midline deviation and the threshold below which they find the deviation ''acceptable. '' (Angle Orthod. 2010;80:515-520.)
Leptin (LEP) is reported to play important roles in controlling energy balance in vertebrates, including birds. However, it remains an open question whether an authentic "LEP gene" exists and functions in birds. Here, we identified and characterized a LEP gene (zebra finch LEP [zbLEP]) encoding a 172-amino acid precursor in zebra finches. Despite zbLEP showing limited amino acid sequence identity (26%-29%) to human and mouse LEPs, synteny analysis proved that zbLEP is orthologous to mammalian LEP. Using a pAH32 luciferase reporter system and Western blot analysis, we demonstrated that the recombinant zbLEP protein could potently activate finch and chicken LEP receptors (zbLEPR; cLEPR) expressed in human embryonic kidney 293 cells and enhance signal transducer and activator of transcription 3 phosphorylation, further indicating that zbLEP is a functional ligand for avian LEPRs. Interestingly, quantitative real-time RT-PCR revealed that zbLEP mRNA is expressed nearly exclusively in the pituitary and various brain regions but undetectable in adipose tissue and liver, whereas zbLEPR mRNA is widely expressed in adult finch tissues examined with abundant expression noted in pituitary, implying that unlike mammalian LEP, finch LEP may not act as an adipocyte-derived signal to control energy balance. As in finches, a LEP highly homologous to zbLEP was also identified in budgerigar genome. Strikingly, finch and budgerigar LEPs show little homology with chicken LEP (cLEP) previously reported, suggesting that the so-called cLEP is incorrect. Collectively, our data provide convincing evidence for the existence of an authentic functional LEP in avian species and suggest an important role of brain- and pituitary-derived LEP played in vertebrates.
Cancer stem cells (CSCs) have been proposed and evidenced as the initiator of tumor formation and the seeds of metastases. Thereby, the molecular mechanisms regarding modulation of CSCs have been widely explored, aimed to improve treatment for cancer patients. Recent progress has highlighted the effects of Hippo signaling in tumorigenesis and cancer development, including its crucial role in CSC regulation. Although the kernel Hippo signaling cascade has been well studied, its upstream inputs and downstream transcriptional regulation still remain elusive. In this review, we summarize the current understanding of the mechanism and regulatory function of Hippo signaling in CSCs, with emphasis on its possible roles in regulation of CSC self-renewal, differentiation and tumorigenesis.
Mechanical stress has been considered to be an important factor in bone remodeling and recent publications have shown that mechanical stress can regulate the direction of stem cell differentiation. The exact mechanobiological effects of pressure on initial osteodifferentiation of mesenchymal stem cells (MSCs) have not been determined. These mechanobiological mechanisms may be important both in biological responses during orthodontic tooth movement and in the development of new mechanobiological strategies for bone tissue engineering. We investigated the effects of static (23 kPa) or dynamic (10-36 kPa and at 0.25 Hz frequency) pressure on MSCs during the initial process of osteoblastic differentiation following treatment with dexamethasone, beta-glycerophosphate and ascorbic acid (for 0, 3, and 7 days, respectively). The following parameters were analyzed in the ALPase activity, mRNA level of osteogenesis-related transcription factors (Runx2, Osterix, Msx2, and Dlx5), and phosphorylation of ERK1/2 and p38 MAPK. The results showed that exposure to either dynamic or static pressure induced initial osteodifferentiation of MSCs. Particularly both types of pressure strongly stimulated the expression of osteogenesis-related factors of totally undifferentiated MSCs. ERK signaling participated in early osteodifferentiation and played a positive but non-critical role in mechanotransduction, whereas p38 MAPK was not involved in this process. Furthermore, the undifferentiated MSCs were highly sensitive to pressure exposure; whereas after osteoinduction MSCs reacted to pressure in a lower response state. The findings should lead to further studies to unveil the complex initial biological mechanisms of bone remodeling and regeneration upon mechanical stimuli.
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