ORIGINAL RESEARCH
Pentoxifylline Attenuates Transforming Growth Factor-β1-Stimulated Elastogenesis in Human Tunica Albuginea-Derived Fibroblasts Part 2: Interference in a TGF-β1/Smad-Dependent Mechanism and Downregulation of AAT1

https://doi.org/10.1111/j.1743-6109.2010.01749.xGet rights and content

ABSTRACT

Introduction

Transforming growth factor-beta1 (TGF-β1) contributes to the pathogenesis of Peyronie's disease (PD). Pentoxifylline (PTX) antagonizes the effects of TGF-β1 and has been utilized in our clinic for the management of PD although the mechanisms of action are not entirely clear.

Aim

We studied cell-signaling pathways through which TGF-β1 and PTX mediate collagen metabolism, elastin expression, and elastogenesis in tunica albuginea-derived fibroblasts (TADFs).

Methods

TADFs from men with and without PD were cultured and treated with TGF-β1 and PTX as monotherapy at differing concentrations and time points. Combination treatment (TGF-β1 followed by PTX and vice versa) was also investigated.

Main Outcome Measures

Reverse-transcription polymerase chain reaction and Western blotting were utilized to assess differences in elastin metabolism and cellular signaling between groups. Alpha-1 antitrypin (AAT1) expression was assayed.

Results

At doses greater than 0.1 ng/Ml, TGF-β1 increased messenger ribonucleic acid (mRNA) and protein expression of elastin in a time-dependent fashion in TADF. PTX did not interfere with TGF-β1 mediated upregulation of elastin mRNA and protein in TADF. However, pretreatment of TADF with PTX was associated with decreased expression of AAT1, decreased activity of the Smad1/5 pathway, and enhanced phosphorylation of the inhibitory Smad6.

Conclusion

Expression of elastin mRNA and protein is upregulated in TADF by TGF-β1. PTX has no effect on elastin production but attenuates elastogenesis in TADF through an AAT1-related mechanism. Lin G, Shindel AW, Banie L, Ning H, Huang YC, Liu G, Lin CS, and Lue TF. Pentoxifylline attenuates TGF-β1-stimulated elastogenesis in human tunica albuginea-derived fibroblasts Part 2: interference in a TGF-β1/Smad-dependent mechanism and downregulation of aat1.

Introduction

Transforming growth factor-beta1 (TGF-β1) is a potent modulator of the ECM in a variety of human fibrotic diseases [1]. It is known to be intimately related to the Peyronie's disease (PD) phenotype, and treatment with TGF-β1 has been shown to induce a PD-like condition in animal models 2, 3, 4. Much of this effect has been attributed to TGF-β1-induced enhancement of collagen production in fibroblasts although alternate pathways mediated by other members of the TGF-β1 superfamily (such as myostatin) have been shown to be involved in the pathophysiology of PD 5, 6, 7. TGF-β1 has also been demonstrated to increase elastin mRNA levels in both dermal and lung fibroblasts 8, 9.

TGF-β1 exerts the majority of its downstream effects via action on Smad proteins, a family of TGF receptor substrates that have the capacity to act as transcription factors in the cell nucleus [1]. Smad proteins frequently interact with one another during the course of activation; specifically, Smad2 and 3 typically interact together, whereas Smad1 and 5 also share activity. Although the majority of Smad proteins have stimulatory effects on gene transcription, the inhibitory Smads (Smad6 and 7) inhibit the activity of other Smad proteins 10, 11. The Smad proteins have been shown to play very important roles in the modulation of collagen I production and thereby a critical role in extracellular fibrotic conditions, such as PD [1].

Pentoxifylline (PTX), a nonselective phosphodiesterase inhibitor with anti-inflammatory properties in vivo and in vitro, has been shown in both in vitro and in vivo (rat) experiments to induce regression of collagen and TGF-β1-induced plaque [12]. We have demonstrated that TGF-β1 enhances collagen and elastin production as well as elastogenesis in tunica albuginea-derived fibroblast cells (TADF). In this model system elastogenesis (deposition of elastin fibers) is attenuated by pretreatment with PTX. The current study is an assessment of the impact of TGF-β1 with or without PTX on elastin metabolism in tunica albuginea derived fibroblasts (TADF) at the mRNA, protein, and cellular signaling level [13].

Section snippets

Tissue Harvesting and Cell Culture

Fibrotic tunica plaques (PT) were harvested from 12 patients with chronic (>12 months duration) PD who were undergoing surgery for correction of penile curvature. Normal tunica (NT) was harvested from six patients who were undergoing penile prosthesis placement. All cavernosal tissue was stripped from the biopsy specimens so as to ensure a pure culture of tunica-derived tissues. Our Institutional Committee on Human Research approved all procedures regarding the collection and use of human

TGF-β1 Stimulates the Synthesis of Elastin in a Time- and Dose-Dependent Manner in TADF Cells

TGF-β1 increased elastin mRNA expression in cultured TADF in a dose-dependent fashion with a plateau effect at doses greater than 0.1 ng/mL for both PT and NT TADF. The magnitude of increase in NT was significantly less than PT at doses greater than 1 ng/mL (Figure 1A). Steady-state elastin mRNA levels were significantly increased in TGF-β1 treated TADF relative to untreated TADF. Increased elastin mRNA was detected within 4 hours in PT cells; interestingly, NT TADF did not significantly increase

Discussion

The TGF-β superfamily binds to two receptors, TβRI and TβRII, to regulate downstream molecules and subsequent gene expression. After the TGF-β1 ligand binds to a receptor, signal transduction occurs predominantly by members of the Smad family. The activation of Smad proteins is achieved through the phosphorylation of specific receptor-regulated Smads (R-Smads) by an activated receptor. This leads to formation of “Smad complexes” that accumulate in the nucleus and regulate target genes 10, 11.

Conclusions

Production of collagen and elastin in TADF cells is stimulated by TGF-β1; this effect is associated with increased activation of the dual TGF-β/Smad pathways. Pretreatment of TADF with PTX attenuates the TGF-β1 mediated increase in elastogenesis, possibly by downregulation of Smad1/5 via an iSmad6 dependent mechanism. As there were no PTX-mediated changes in expression/activity of the proteins essential to assembly of elastin fibers in the ECM, the precise mechanisms by which PTX attenuates

Category 1

  • (a)

    Conception and Design

    Guiting Lin; Tom F. Lue; Ching-Shwun Lin

  • (b)

    Acquisition of Data

    Guiting Lin; Hongxiu Ning; Lia Banie; Gang Liu; Yun-Ching Huang

  • (c)

    Analysis and Interpretation of Data

    Guiting Lin; Alan W. Shindel; Ching-Shwun Lin; Tom F. Lue

Category 2

  • (a)

    Drafting the Article

    Alan W. Shindel

  • (b)

    Revising It for Intellectual Content

    Guiting Lin; Tom F. Lue; Alan W. Shindel; Ching-Shwun Lin

Category 3

  • (a)

    Final Approval of the Completed Article

    Guiting Lin; Tom F. Lue; Alan W. Shindel

References (35)

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Conflict of Interest: None.

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