A heterocyclic molecule kartogenin induces collagen synthesis of human dermal fibroblasts by activating the smad4/smad5 pathway

Highlights

• A small novel molecule KGN is found to stimulate type-I collagen synthesis of fibroblasts.

• KGN has no obvious cytotoxicity on fibroblasts’ viability, morphology and survival.

• Smad4/smad5 signaling pathway is activated by KGN.

Abstract

Declined production of collagen by fibroblasts is one of the major causes of aging appearance. However, only few of compounds found in cosmetic products are able to directly increase collagen synthesis. A novel small heterocyclic compound called kartogenin (KGN) was found to stimulate collagen synthesis of mesenchymal stem cells (MSCs). So, we hypothesized and tested that if KGN could be applied to stimulate the collagen synthesis of fibroblasts.

Human dermal fibroblasts in vitro were treated with various concentrations of KGN, with dimethyl sulfoxide (DMSO) serving as the negative control. Real-time reverse-transcription polymerase chain reaction, Western blot, and immunofluorescence analyses were performed to examine the expression of collagen and transforming growth factor beta (TGF-β) signaling pathway. The production of collagen was also tested in vivo by Masson’s trichrome stain and immunohistochemistry in the dermis of mice administrated with KGN.

Results showed that without obvious influence on fibroblasts’ apoptosis and viability, KGN stimulated type-I collagen synthesis of fibroblasts at the mRNA and protein levels in a time-dependent manner, but KGN did not induce expression of α-skeletal muscle actin (α-sma) or matrix metallopeptidase1 (MMP1), MMP9 in vitro. Smad4/smad5 of the TGF-β signaling pathway was activated by KGN while MAPK signaling pathway remained unchanged. KGN also increased type-I collagen synthesis in the dermis of BALB/C mice.

Our results indicated that KGN promoted the type-I collagen synthesis of dermal fibroblasts in vitro and in the dermis of mice through activation of the smad4/smad5 pathway. This molecule could be used in wound healing, tissue engineering of fibroblasts, or aesthetic and reconstructive procedures.

Introduction

Collagen is the major structural component of the dermis. Collagen and extracellular matrix degradation accounts for aging of the skin[1]. Collagen, bovine-derived or human-derived has been recognized as a well-accepted treatment for cosmetic purposes since the 1970s [2]. While collagen [3] and analogous composition filling [4] are the main methods for esthetic surgery, biologic and synthetic collagen-embedded wound dressings are used as skin substitutes [5]. During wound healing phases, the formation of collagen-rich granulation tissue is vital for wound closure [6]. For decades, the logic of filling defects by the corresponding collagen components or fibroblasts transplantation [7], [8] is the no-alternative choice for clinical researches.

However, in recent years, an unprecedented approach is to regulate the phenotype of endogenous cells by direct in vivo modulation using drug-like small molecules [9], [10], [11], [12]. The use of unbiased phenotypic or pathway-based high-throughput cellular screens of chemical libraries has identified molecules that affect adult stem cell-related processes, such as inhibiting the signaling pathway of tumor and adult stem cells manipulation [13], [14]. As the major cellular components in the dermis, could fibroblasts produce more collagen by the application of exogenic small molecules? No related molecules have been reported in literature.

In 2012, a heterocyclic compound called kartogenin (KGN) was discovered to stimulate collagen synthesis of mesenchymal stem cells (MSCs) [15]. When KGN bonded with Filamin A (FLNA), KGN disrupted FLNA’s interaction with the transcription factor core-binding factor beta subunit (CBFβ). This study enlightened the future of small molecule-based approaches to adult stem cell therapies. Specific cell populations with variable mesenchymal differentiation potential were obtained from freshly separated and adherent cultured dermal fibroblasts. Various studies proved the fibroblasts had the capacity to differentiate into multiple cell lineages[16], [17], [18]. Given the phenotypic similarity of MSCs and dermal fibroblasts [19], [20], [21], we wonder what effect could KGN have on human dermal fibroblasts? As such, we proposed and tested the hypothesis that the small molecule KGN could stimulate collagen synthesis in human dermal fibroblasts in vitro, and then we examined its effect in the dermis of mice. We further investigated the potential biochemical mechanism of how KGN activated collagen expression in fibroblasts. If these mechanisms were observed, the novel small molecule KGN could be applied conveniently in tissue engineering of fibroblasts, accelerating wound healing, or esthetic and reconstructive skin rejuvenation, instead of expansive tissue components (collagen) filling or complex cell transplantation.