Testosterone regulates keratin 33B expression in rat penis growth through androgen receptor signaling

Abstract

Androgen therapy is the mainstay of treatment for the hypogonadotropic hypogonadal micropenis because it obviously enhances penis growth in prepubescent microphallic patients. However, the molecular mechanisms of androgen treatment leading to penis growth are still largely unknown. To clarify this well-known phenomenon, we successfully generated a castrated male Sprague Dawley rat model at puberty followed by testosterone administration. Interestingly, compared with the control group, testosterone treatment stimulated a dose-dependent increase of penis weight, length, and width in castrated rats accompanied with a dramatic recovery of the pathological changes of the penis. Mechanistically, testosterone administration substantially increased the expression of androgen receptor (AR) protein. Increased AR protein in the penis could subsequently initiate transcription of its target genes, including keratin 33B (Krt33b). Importantly, we demonstrated that KRT33B is generally expressed in the rat penis and that most KRT33B expression is cytoplasmic. Furthermore, AR could directly modulate its expression by binding to a putative androgen response element sequence of the Krt33b promoter. Overall, this study reveals a novel mechanism facilitating penis growth after testosterone treatment in precastrated prepubescent animals, in which androgen enhances the expression of AR protein as well as its target genes, such as Krt33b.

Figure 1

Effects of androgen on rat penis development. (a) Gross morphology of the rat penis by light microscopy with or without testosterone treatment. (b) Difference of the rat penis weight, length, and width after testosterone treatment. Values in each column that do not share the same letter indicate statistical significance; P < 0.05. (c) Histopathological assessment by H and E staining as indicated; *represents a blood vessel, ^▲represents interstitial fibrosis. Scale bars = 100 μm. (d) Masson's trichrome staining of rat cavernous tissue. Smooth muscle and interstitial fibrotic tissues are stained in red and blue, respectively. Scale bars = 50 μm. (e) Quantitative changes in smooth muscle and interstitial fibrotic tissue contents were analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA); n = 4 for each group. *P < 0.05.

Figure 2

Ligand-dependent androgen receptor (AR) protein expression in the rat penis. Protein or mRNA levels of AR in the rat penis (n = 3–4 for each group) was determined using western blotting (a) or RT-PCR (b) with β-actin as an internal control, respectively; images are representative of three separate western blotting and PCR experiments. Quantitative analyses were carried out, and error bars represent mean ± standard error of the mean values in each column that do not share the same letter indicate statistical significance. P < 0.05. (c) Detection of AR protein expression in rat penis tissues by IIF. Cross cryostat sections of a penis probed with an anti-AR antibody (red) and DAPI (blue). Scale bars = 100 μm.

Figure 3

Differential gene expression of Krt33b in the mouse penis. (a) Relative gene expression of the Krt33b gene in the penis of mice at puberty and adulthood based on a gene expression microarray. (b) RT-PCR was carried out to confirm the changes of the Krt33b gene in the penis of mice at puberty and adulthood.

Figure 4

Co-expression of androgen receptor (AR) and keratin 33B (KRT33B) proteins in the rat penis by indirect immunofluorescence staining. Cross cryostat sections of a penis probed with anti-KRT33B antibody (red) and anti-AR antibody (green). Scale bars = 100 μm.

Figure 5

Effects of androgen/AR on Krt33b expression. The levels of KRT33B after testosterone (a) or flutamide (b) administration were determined using western blotting with β-actin as an internal control; the levels of KRT33B in HepG2 (c) and MHCC-97L (d) cells at 48 h after transient transfection of AR siRNA were determined using western blotting with β-actin as an internal control; western blots are representative of three independent experiments. *P < 0.05 (NC vs siAR in each group).

Figure 6

Androgen response elements (AREs) in the Krt33b promoter by a chromatin immunoprecipitation (ChIP) assay. (a) Framework of the human Krt33b gene. The predicted ARE sequence was located at the region from −2368 to −2354 upstream of the TATA box. ARE primers were designed according to this framework, −2389 and −2232 represent the initial sites of the forward and reverse primers, respectively. (b, c) ChIPs were performed using digested chromatin from HepG2 and MHCC-97L cells co-expressing androgen receptor and KRT33B. Purified DNAs were analyzed by standard PCR methods using SimpleChIP Human RPL30 Primers and ARE primers. PCR products were observed for each primer set in the input sample and various ChIP samples, but not in the normal IgG ChIP sample.