J Vet Sci.  2009 Jun;10(2):105-113. 10.4142/jvs.2009.10.2.105.

Low dietary inorganic phosphate affects the lung growth of developing mice

• Xu CX
• Jin H
• Chung YS
• Shin JY
• Hwang SK
• Kwon JT
• Park SJ
• Lee ES
• Minai-Tehrani A
• Chang SH
• Woo MA
• Noh MS
• An GH
• Lee KH
• Cho MH

• KMID: 1102966
• DOI: http://doi.org/10.4142/jvs.2009.10.2.105

Abstract

Inorganic phosphate (Pi) plays a critical role in diverse cellular functions, and regulating the Pi balance is accomplished by sodium-dependent Pi co-transporter (NPT). Pulmonary NPT has recently been identified in mammalian lungs. However, to date, many of the studies that have involved Pi have mainly focused on its effect on bone and kidney. Therefore, current study was performed to discover the potential effects of low Pi on the lung of developing transgenic mice expressing the renilla/firefly luciferase dual reporter gene. Two-weeks old male mice divided into 2 groups and these groups were fed either a low PI diet or a normal control diet (normal: 0.5% Pi, low: 0.1% Pi) for 4 weeks. After 4 weeks of the diet, all the mice were sacrificed. Their lungs were harvested and analyzed by performing luciferase assay, Western blotting, kinase assay and immunohistochemistry. Our results demonstrate that low Pi affects the lungs of developing mice by disturbing protein translation, the cell cycle and the expression of fibroblast growth factor-2. These results suggest that optimally regulating Pi consumption may be important to maintain health.

Keyword

Akt; fibroblast growth factor; inorganic phosphate; lung

MeSH Terms

Animals
Blotting, Western
Carrier Proteins/metabolism
Immunohistochemistry
Lung/drug effects/enzymology/*growth & development/metabolism
Male
Mice
Mice, Transgenic
Phosphoproteins/metabolism
Phosphorus, Dietary/*administration & dosage
Phosphorylation
Phosphotransferases (Alcohol Group Acceptor)/metabolism
Proto-Oncogene Proteins c-akt/metabolism
Sodium-Phosphate Cotransporter Proteins, Type IIa/*metabolism

Figure

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  Fig. 1 Western blot analysis of NPT2b protein in the lungs of mice that were fed a low inorganic phosphate (Pi) diet (0.1% Pi) or a normal (0.5% Pi) diet for 4 weeks. (A) The expression of NPT2b protein. (B) The bands-of-interests were further analyzed by using a densitometer. *p values < 0.05 showed a significant difference (mean ± SE, n = 4).

• 

  Fig. 2 Western blot analysis of the Akt and phospho-Akt protein in the lungs of mice fed a low Pi diet (0.1% Pi) or a normal (0.5% Pi) diet for 4 weeks. (A) The expressions of Akt and phospho-Akt protein in the lungs. (C) The bands-of-interests were further analyzed by using a densitometer. (B) The Akt kinase activity was measured in the lung homogenates. *p values < 0.05 showed a significant difference compared with normal (mean ± SE, n = 4).

• 

  Fig. 3 Western blot analysis of the mammalian target of rapamycin (mTOR), 4E-PB1 and p-4E-BP1 protein in the lungs of mice fed a low Pi diet (0.1% Pi) or a normal (0.5% Pi) diet for 4 weeks. (A) The expressions of mTOR, 4E-PB1 and p-4E-BP1 protein in the lungs. (B) The mTOR kinase activity and phosphorylation ratio for 4E-BP1 were measured in the lung homogenates. (C) The bands-of-interests were further analyzed by using a densitometer. (D) The luciferase activities were measured in the tissue homogenate from lung, and the ratios of the cap-dependent (r-luc) to the IRES dependent (f-luc) protein translation are shown. p values (*p < 0.05, **p < 0.01) indicate a significant difference compared with normal (mean ± SE, n = 4).

• 

  Fig. 4 Western blot analysis of the cell cycle signaling proteins. The lungs of mice fed a low Pi diet (0.1% Pi) or a normal (0.5% Pi) diet for 4 weeks. (A) The expressions of p53, p21 and p27 protein in lung. (B) The expressions of cyclin D3, cyclin-dependent kinase 4 (CDK4) and proliferating cell nuclear antigen (PCNA) protein in lung. (C, D) The bands-of-interests were further analyzed by using a densitometer. (E) Immunohistochemical measurement of PCNA in the lung. The dark brown color indicates the PCNA expression (scale bar = 100 µm). (F) Comparison of the PCNA labeling index in the lungs. p values (*p < 0.05, **p < 0.01) indicate a significant difference compared with normal (mean ± SE, n = 4).

• 

  Fig. 5 Analysis of fibroblast growth factor 2 (FGF-2) protein in the lungs of mice fed a low Pi diet (0.1% Pi) or a normal (0.5% Pi) diet for 4 weeks. (A) The expression of FGF-2 protein in the lung. (B) The bands-of-interests were further analyzed by using a densitometer. (C) Immunohistochemical measurement of FGF-2 in the lung of transgenic mice. The dark brown color indicates the expression of FGF-2 (scale bar = 100 µm). (D) Comparison of the FGF-2 labeling index in the lungs. p values (*p < 0.05, **p < 0.01) indicate a significant difference compared with normal (mean ± SE, n = 4).

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