The role of hedgehog-interacting protein in maintaining cavernous nerve integrity and adult penile morphology

Abstract

Introduction: Sonic hedgehog (SHH) is an essential regulator of smooth muscle apoptosis in the penis that has significant clinical potential as a therapy to suppress post-prostatectomy apoptosis, an underlying cause of erectile dysfunction (ED). Thus an understanding of how SHH signaling is regulated in the adult penis is essential to move the field of ED research forward and to develop new treatment strategies. We propose that hedgehog-interacting protein (HIP), which has been shown to bind SHH protein and to play a role in SHH regulation during embryogenesis of other organs, is a critical regulator of SHH signaling, penile morphology, and apoptosis induction.

Aims: We have examined HIP signaling in the penis and cavernous nerve (CN) during postnatal differentiation of the penis, in CN-injured, and a diabetic model of ED.

Methods: HIP localization/abundance and RNA abundance were examined by immunohistochemical (IHC) analysis and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in Sprague-Dawley rats between the ages of 7 and 92 days old, in CN-injured Sprague-Dawley rats and in BioBreeding/Worcester diabetic rats. HIP signaling was perturbed in the pelvic ganglia and in the penis and TUNEL assay was performed in the penis. CN tie, lidocaine, and anti-kinesin experiments were performed to examine HIP signaling in the CN and penis.

Results: In this study we are the first to demonstrate that HIP undergoes anterograde transport to the penis via the CN, that HIP perturbation in the pelvic ganglia or the penis induces apoptosis, and that HIP plays a role in maintaining CN integrity, penile morphology, and SHH abundance.

Conclusions: These studies are significant because they show HIP involvement in cross-talk (signaling) between the pelvic ganglia and penis, which is integral for maintenance of penile morphology and they suggest a mechanism of how nerves may regulate target organ morphology and function.

Figure 1

Diagram of Sonic hedgehog, patched (PTCH1), smoothened (SMO), and hedgehog-interacting protein (HIP) interactions. S = SHH; P = PTCH1.

Figure 2

Immunohistochemical analysis of hedgehog-interacting protein (HIP) in the dorsal nerve bundle (A) and in the corpora cavernosa (B) of the penis of P7-P92 Sprague-Dawley rats. (a) HIP protein is localized in the nerves of the penis at all ages assayed. An unusual ultrastructure was observed in the nerves at P22 and P42. X250. (B) HIP was observed in a layer under the tunica at P7 (×1,000). HIP localization was restricted to the developing sinuses of the corpora cavernosa and individual cells between the sinuses that are likely fibroblasts at P12–P22 (×1,000) and at P42–P92 (×400). At P12, P22, and P42, intense HIP staining was observed in small structures within and between the developing smooth muscle cells. Arrows indicate HIP staining.

Figure 3

Quantification of Hip RNA and localization of the hedgehog-interacting protein (HIP) in penis tissue. (A) Time course of Hip RNA expression quantified by real-time reverse transcriptase-polymerase chain reaction in Sprague-Dawley rat penises. Hip expression was most abundant during the first week after birth. Hip expression remained constant but low by comparison in the adult penis. A small spike in Hip expression was observed with the onset of puberty (P40-P60). (B) Confocal microscopy of normal Sprague-Dawley rat penises assayed for HIP protein. HIP was abundant in the nucleus and cytoplasm of endothelial cells and in the cellular membrane and cytoplasm of smooth muscle cells of the corpora cavernosal sinusoidal tissue. (C) Dual ACTA1/HIP IHC confirms the presence of HIP protein in penile smooth muscle of control and 2 days cavernous nerve-cut rats. E = endothelium; SM = smooth muscle.

Figure 4

Quantification of Hip, Shh, and Ptch1 RNA and hedgehog-interacting protein (HIP) in cavernous nerve (CN)-cut rats. (A) Quantification of SHH, PTCH1, and HIP proteins in control, 2 and 5 days CN-injured Sprague-Dawley rat penises. SHH and PTCH1 proteins were significantly decreased at 2 and 5 days after CN injury. HIP was unchanged at 2 days after CN injury but was significantly decreased 1.4-fold 5 days after CN injury. (B) Hip RNA was quantified by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) using Rpl19 as an internal standard. There was no change in Hip expression at 2 days after CN injury however Hip expression was significantly increased twofold at 5 days after CN injury (P value = 0.03). (C) Shh RNA was quantified by real-time RT-PCR using Rpl19 as an internal standard. Shh expression significantly increased 2.4 and 1.7-fold at 2 and 5 days after CN injury (P values = 0.001 and 0.002). (D) Ptch1 RNA was quantified by real-time RT-PCR using Rpl19 as an internal standard. Ptch1 expression significantly increased 4.6- and 2.3-fold at 2 and 5 days after CN injury (P values = 0.05 and 0.03).

Figure 5

Affi-Gel beads soaked in hedgehog-interacting protein (HIP) inhibitor, HIP protein, and phosphate-buffered saline (control) were implanted under the pelvic ganglia of Sprague-Dawley rats and TUNEL, confocal microscopy, and HIP protein quantification were performed in the penis. (A) TUNEL assay showed that apoptosis was increased 2.4-fold in the corpora cavernosa after 2 days of HIP inhibition in the pelvic ganglia and 1.5-fold in the corpora cavernosa after HIP protein treatment of the pelvic ganglia. (B) Verification of HIP inhibitor function in vivo in the corpora cavernosa was performed by staining the HIP inhibited penis tissue with a second HIP antibody (Abcam ab39208). As the HIP inhibitor was already bound to HIP in the corpora cavernosal tissue, the Abcam antibody should not stain in the region of the Affi-Gel beads; however, staining should be present in untreated tissue away from the beads as was observed. Arrows indicate HIP protein. (X160). (C) Electron microscopy (EM) of HIP inhibited corpora cavernosa showed much less smooth muscle abundance by comparison with controls (×44,000) and endothelial apoptosis was evident (×70,000). (D) EM of HIP inhibited CN (×30,000) showed axonal degeneration and demyelination of nerve fibers by comparison with controls (×44,000). Arrows indicate degenerating axons. (E) HIP protein in the corpora cavernosa remained unchanged at 2 days of HIP inhibition in the pelvic ganglia but was significantly decreased 1.4-fold after 5 days of HIP inhibition in the pelvic ganglia. TUNEL assay showed that apoptosis was increased 2.4-fold in the corpora cavernosa after 2 days of HIP inhibition directly in the penis and 2.2-fold in the corpora cavernosa after direct HIP protein treatment of the penis via Affi-Gel beads. B = Affi-Gel bead; E = endothelium; SM = smooth muscle.

Figure 6

Hedgehog-interacting protein (HIP) localization and quantification in the cavernous nerve (CN) tie, lidocaine, anti-kinesin-treated Sprague-Dawley rats, and in diabetic rats. (A) A tie was placed on the CNs in order to determine if HIP protein was under going anterograde transport to the penis via the CN. HIP protein built up on the ganglia side of the tie after 2 days (left and middle, ×100, and right, ×250) showing that HIP protein was transported by the CN. HIP protein was observed originating in neurons of the pelvic ganglia (Right, ×160). (B) HIP protein was significantly decreased 1.2-fold in corpora cavernosa of rats in which their CN’s were treated with lidocaine. (C) HIP was significantly decreased 1.3-fold in corpora cavernosa of rats in which their CN’s were treated with anti-kinesin. (D) HIP protein was significantly decreased 1.2-fold in diabetic penises and Hip RNA expression was increased 3.1-fold in diabetic penises.