1. Volume 93, Issue 1, Pages 54-68 (January 2018)
Transcription factor MafB may play an important role in secondary hyperparathyroidism 
Naoki Morito, Keigyou Yoh, Toshiaki Usui, Hisashi Oishi, Masami Ojima, Akiko Fujita, Ryusuke Koshida, Hossam H. Shawki, Michito Hamada, Masafumi Muratani, Kunihiro Yamagata, Satoru Takahashi 
Kidney International 
Volume 93, Issue 1, Pages (January 2018)
DOI: /j.kint
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2. Figure 1
MafB expression in the adult parathyroid gland. Immunohistochemical examination revealed that CaSR (Calcium-Sensing Receptor)-positive (red) parathyroid cells also expressed green fluorescence protein (GFP) (green) in the MafB-GFP knockin (MafB+/–) mouse. This fact indicates that MafB is expressed in the adult parathyroid gland. Bar = 50 μm. To optimize viewing of this image, please see the online version of this article at
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3. Figure 2
MafB haploinsufficiency does not affect renal function in mice fed an adenine-supplemented diet. (a) Diagram of chronic kidney disease induction by adenine feeding. Six-week-old wild-type (WT) and MafB+/– mice were fed either normal or adenine-supplemented chow for 6 weeks. (b) The blood urea nitrogen (BUN) and serum creatinine levels after adenine feedings in MafB+/– mice were similar to those in WT mice. n = 10 mice per group. Each bar represents the mean ± SD. **P < (c) Representative renal histology at 12 weeks old. Periodic acid-Schiff stain. Bar = 200 μm. To optimize viewing of this image, please see the online version of this article at
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4. Figure 3
Suppression of secondary hyperparathyoidism in MafB+/– mice fed an adenine-supplemented chow. (a) Serum analysis of calcium, phosphate, intact parathyroid hormone (PTH), and Fgf23. The serum calcium and intact PTH levels were significantly lower in adenine-fed MafB+/– mice compared with wild-type (WT) controls. n = 10 mice per group. *P < 0.05, **P < (b) Parathyroid gland size in WT and MafB+/– mice fed with regular or adenine-supplemented chow. Bars = 100 μm. (c) Cross-sectional areas were measured using hematoxylin and eosin–stained sections and corrected for body weight (μm2/g body weight). The surface area, the cell profile number, and the mean cell surface area of the parathyroid glands were increased in both WT and MafB+/– mice fed adenine-supplemented chow. However, the surface area and the cell profile number increase were blunted somewhat in MafB+/– mice, but the mean cellular size was not. (d) Parathyroid proliferation rates as assessed by Ki-67 immunostaining. (e) Ki-67 proliferation rate was calculated as Ki-67–positive cells/total number of cells. The proliferation rate of parathyroids in MafB+/– mice fed adenine-supplemented chow was significantly lower than that of WT mice fed the same diet. Bars = 30 μm. n = 4–6 mice per group. *P < 0.05, **P < To optimize viewing of this image, please see the online version of this article at
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5. Figure 4
Upregulation of PTH and Ccnd2 expression in parathyroid/thyroid tissue in response to being fed an adenine-supplemented chow is suppressed in MafB+/– mice. (a) Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of PTH, MafB, Ccnd1 (Cyclin D1), Ccnd2 (Cyclin D2), and Kl (Klotho) expression in parathyroid/thyroid tissue. Increased expressions of PTH and MafB in the parathyroid/thyroid tissue was suppressed in MafB+/– mice. Expression of Ccnd1 and Ccnd2 was increased in wild-type (WT) mice fed an adenine-supplemented chow. However, the increase in expression of Ccnd2 was greatly reduced in MafB+/– mice. There were no apparent differences between Ccnd1 expression in WT and MafB+/– parathyroid/thyroid tissue in response to being fed an adenine-supplemented chow. Expression of Kl was significantly suppressed in both WT and MafB+/– mice fed adenine-supplemented chow. However, there was no significant difference between Kl expression in WT and MafB+/– mice fed control or adenine-supplemented chow. Immunohistochemical (IHC) analysis of Ccnd1 (b) and Ccnd2 (c) expression. Bar = 20 μm. Graph depicting semiquantitative analysis by IHC. Relative fluorescence intensity was calculated using the mean fluorescence intensity of WT mice fed a normal diet defined as 1.0. Consistent with RT-PCR analysis, the elevation of Ccnd2 was reduced in the parathyroid/thyroid tissue of MafB+/– mice fed an adenine-supplemented chow relative to WT mice fed the same diet. Values represent the means ± SD, n = 4–5 mice per group. *P < 0.05, **P < To optimize viewing of this image, please see the online version of this article at
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6. Figure 5
Parathyroid hormone (PTH) secretion in response to ethylene glycol tetraacetic acid (EGTA) administration is impaired in MafB+/– mice. Hypocalcemia was induced by EGTA administration. Serum calcium and intact PTH were measured sequentially (0, 0.5, 1, 2, and 4 hours). (a) Hypocalcemia was prolonged in MafB+/– mice. (b) Serum intact PTH was decreased in MafB+/– animals. Quantitative reverse-transcriptase polymerase chain reaction assay of PTH (c) and MafB (d) in parathyroid/thyroid 4 hours after injection of EGTA. Expression of PTH and MafB is induced by hypocalcemia. This increase was blunted in MafB+/– mice. Values represent the means ± SD, n = 5 mice per group. *P < 0.05, **P < 0.01.
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7. Figure 6
MafB regulates transcription of PTH and Ccnd2. (a) A MafB binding site (Maf recognition element [MARE]) that is highly conserved between mouse and human in the evolutionally conserved region 4 of PTH. (b) Reporter assay. Wild-type MARE (WT-Luc) and mutated MARE (Mut-Luc) luciferase reporter constructs are indicated. NIH 3T3 cells were cotransfected with the indicated PTH promoter–reporter plasmid constructs along with the MafB expression plasmid, and the relative luciferase activity was measured as described in the Methods. (c) The relative luciferase activity of the reporter plasmids is shown with the activity generated from cells transfected with the empty reporter vector (0 ng) and the MafB expression plasmid defined as 1.0. (d) A MARE conserved between the promoter region of mouse and human Ccnd2. (e) Reporter assay of Ccnd2. WT MARE (WT-Luc) and mutated MARE (Mut-Luc) luciferase reporter constructs are indicated. (f) The relative luciferase activity of the reporter plasmids is shown. Each bar represents the mean ± SD. **P < 0.05.
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8. Figure 7
Identification of MafB targets in the parathyroid gland. (a) Diagram showing the genes identified by RNA-sequence (RNA-seq) analysis. RNA samples prepared from the MafB GFP/− knockout and MafBGFP/+ control parathyroid glands were subjected to RNA-seq analysis. (b) List of the 197 genes as MafB targets (85 genes were upregulated and 112 genes were downregulated). *Mitochondrial genes. The underlined genes are known to be expressed specifically in the parathyroid. (c) Dysregulated signaling pathways in the absence of MafB. The top 10 deregulated Reactome pathways ranked by P values. This analysis was conducted with REACTOME ( n = 3 mice per group.
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9. Figure 8
RNA-sequencing (RNA-seq) read mapping revealed severe depletion of MafB, Gata3, and Gcm2 expression in MafB GFP/− knockout (KO) parathyroid glands. (a) RNA-seq read mapping on the loci of 6 parathyroid-specific genes. Representative images are shown. RNA-seq data were visualized on the UCSC Genome Browser ( (b) Significant (P < 0.05) depletion of MafB, Gata3, Gcm2, and Kl was observed in MafB GFP/− KO mice. The depletion of PTH was also observed, but was not significant (P = 0.117). Egr-1 was elevated significantly in MafBGFP/− KO mice. Transcript expression values were determined after transcript normalization (reads per kilobase per million [RPKM]). n = 3 mice per group.
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10. Figure 9
Twelve weeks inactivation of MafB did not affect parathyroid gland size or intact parathyroid hormone (PTH) levels under normal conditions. (a) Experimental time course of tamoxifen-induced global MafB deletion. (b) Quantitative reverse-transcriptase polymerase chain reation analysis of MafB expression in parathyroid/thyroid tissue. MafB mRNA level was less than 10% of control mice at 4 and 12 weeks after tamoxifen injection in the knockout (KO) mice. (c) There were no significant differences in blood urea nitrogen (BUN) and serum creatinine between MafB KO and control mice at 12 weeks after tamoxifen injection. (d) Histology of parathyroid gland at 12 weeks after tamoxifen injection. There was no significant difference in parathyroid gland size between MafB KO and MafBflox/flox (control) mice. Hematoxylin and eosin stain. Bars = 100 μm. (e) Serum analysis of calcium, phosphate, and intact PTH. There were no significant differences in calcium, phosphate, and intact PTH between MafB KO and control mice at 12 weeks after tamoxifen injection. Values represent the means ± SD, n = 10 mice per group. **P < BW, body weight. To optimize viewing of this image, please see the online version of this article at
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11. Figure 10
Parathyroid hormone (PTH) secretion in response to hypocalcemia was blunted in MafB knockout (KO) mice. (a) Hypocalcemia, induced by ethylene glycol tetraacetic acid (EGTA), was prolonged in MafB KO mice. (b) Serum intact PTH elevation in response to hypocalcemia was blunted in MafB KO mice. (c) PTH mRNA expression 4 hours after hypocalcemia induction was suppressed in MafB KO mice. (d) MafB mRNA expression 4 hours after hypocalcemia induction was increased in both control and MafB KO mice. However, the expression in KO mice was approximately 10% of control mice. Values represent the means ± SD, n = 5 mice per group. *P < 0.05, **P < 0.01.
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12. Figure S1
MafB knockout (KO) mice developed renal insufficiency 16 weeks after tamoxifen injection. (A) Blood urea nitrogen (BUN) and serum creatinine were increased in MafB KO mice compared with MafBflox/flox mice at 16 weeks after tamoxifen injection, but only the increase in BUN was significant (P < 0.05) (serum creatinine, P = 0.09). (B) Serum analysis of calcium, phosphate, and intact parathyroid hormone (PTH). There was no significant difference in serum calcium, phosphate, and intact PTH between MafB KO and MafBflox/flox (control) mice. n = 7 mice per group. Each bar represents the mean ± SD. *P < 0.05.
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