Hee-Jae Cha

The Role of Thymosin Beta 4 on Odontogenic Differentiation in Human Dental Pulp Cells

We recently reported that overexpression of thymosin beta-4 (Tβ4) in transgenic mice promotes abnormal hair growth and tooth development, but the role of Tβ4 in dental pulp regeneration was not completely understood. The aim of this study was to investigate the role of Tβ4 on odontoblastic differentiation and the underlying mechanism regulating pulp regeneration in human dental pulp cells (HDPCs). Our results demonstrate that mRNA and protein expression of Tβ4 is upregulated during odontogenic differentiation in HDPCs. Transfection with Tβ4 siRNA decreases OM-induced odontoblastic differentiation by decreasing alkaline phosphatase (ALP) activity, mRNA expression of differentiation markers, and calcium nodule formation. In contrast, Tβ4 activation with a Tβ4 peptide promotes these processes by enhancing the phosphorylation of p38, JNK, and ERK mitogen-activated protein kinases (MAPKs), bone morphogenetic protein (BMP) 2, BMP4, phosphorylation of Smad1/5/8 and Smad2/3, and expression of transcriptional factors such as Runx2 and Osterix, which were blocked by the BMP inhibitor noggin. The expression of integrin receptors α1, α2, α3, and β1 and downstream signaling molecules including phosphorylated focal adhesion kinase (p-FAK), p-paxillin, and integrin-linked kinase (ILK) were increased by Tβ4 peptide in HDPCs. ILK siRNA blocked Tβ4-induced odontoblastic differentiation and activation of the BMP and MAPK transcription factor pathways in HDPCs. In conclusion, this study demonstrates for the first time that Tβ4 plays a key role in odontoblastic differentiation of HDPCs and activation of Tβ4 could provide a novel mechanism for regenerative endodontics.

Evaluation of Skeletal and Cardiac Muscle Function after Chronic Administration of Thymosin β-4 in the Dystrophin Deficient Mouse

Thymosin beta-4 (Tβ4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. We studied the effects of chronic administration of Tβ4 on the skeletal and cardiac muscle of dystrophin deficient mdx mice, the mouse model of Duchenne muscular dystrophy. Female wild type (C57BL10/ScSnJ) and mdx mice, 8–10 weeks old, were treated with 150 µg of Tβ4 twice a week for 6 months. To promote muscle pathology, mice were exercised for 30 minutes twice a week. Skeletal and cardiac muscle function were assessed via grip strength and high frequency echocardiography. Localization of Tβ4 and amount of fibrosis were quantified using immunohistochemistry and Gomoris tri-chrome staining, respectively. Mdx mice treated with Tβ4 showed a significant increase in skeletal muscle regenerating fibers compared to untreated mdx mice. Tβ4 stained exclusively in the regenerating fibers of mdx mice. Although untreated mdx mice had significantly decreased skeletal muscle strength compared to untreated wild type, there were no significant improvements in mdx mice after treatment. Systolic cardiac function, measured as percent shortening fraction, was decreased in untreated mdx mice compared to untreated wild type and there was no significant difference after treatment in mdx mice. Skeletal and cardiac muscle fibrosis were also significantly increased in untreated mdx mice compared to wild type, but there was no significant improvement in treated mdx mice. In exercised dystrophin deficient mice, chronic administration of Tβ4 increased the number of regenerating fibers in skeletal muscle and could have a potential role in treatment of skeletal muscle disease in Duchenne muscular dystrophy.

Hepatic Stellate Cells Express Thymosin Beta 4 in Chronically Damaged Liver

Although the various biological roles of thymosin β4 (Tβ4) have been studied widely, the effect of Tβ4 and Tβ4-expressing cells in the liver remains unclear. Therefore, we investigated the expression and function of Tβ4 in chronically damaged livers. CCl4 was injected into male mice to induce a model of chronic liver disease. Mice were sacrificed at 6 and 10 weeks after CCl4 treatment, and the livers were collected for biochemical analysis. The activated LX-2, human hepatic stellate cell (HSC) line, were transfected with Tβ4-specific siRNA and activation markers of HSCs were examined. Compared to HepG2, higher expression of Tβ4 in RNA and protein levels was detected in the activated LX-2. In addition, Tβ4 was up-regulated in human liver with advanced liver fibrosis. The expression of Tβ4 increased during mouse HSC activation. Tβ4 was also up-regulated and Tβ4-positive cells were co-localized with α-smooth muscle actin (α-SMA) in the livers of CCl4-treated mice, whereas such cells were rarely detected in the livers of corn-oil treated mice. The suppression of Tβ4 in LX-2 cells by siRNA induced the down-regulation of HSC activation-related genes, tgf-β, α-sma, collagen, and vimentin, and up-regulation of HSC inactivation markers, ppar-γ and gfap. Immunofluorescent staining detected rare co-expressing cells with Tβ4 and α-SMA in Tβ4 siRNA-transfected cells. In addition, cytoplasmic lipid droplets were observed in Tβ4 siRNA-treated cells. These results demonstrate that activated HSCs expressed Tβ4 in chronically damaged livers, and this endogenous expression of Tβ4 influenced HSC activation, indicating that Tβ4 might contribute to liver fibrosis by regulating HSC activation.

Analysis of Expression Patterns of Thymosin β4 and CD133 in Normal Stomach

Thymosin β4 (Tβ4) has been reported to be overexpressed in CD133-positive colorectal cancer stem cells. We analyzed the relationship between Tβ4 and CD133-positive stem cells in normal stomach by examining the expression patterns of Tβ4 and CD133 in normal stomach tissues by immunohistochemical staining; co-localization of Tβ4 and CD133 was studied by immunofluorescence and confocal laser-scanning microscopy. Both Tβ4 and CD133 were expressed in stomach glands and showed similar expression patterns. Immunofluorescence staining of Tβ4 and CD133 showed that the expression of Tβ4 and CD133 was co-localized. In summary, both Tβ4 and CD133 were expressed in glands of normal stomachs and expression patterns were co-localized. These data suggest that Tβ4 expression is strongly related to CD133 expression.

Expression of thymosin beta-4 in human periodontal ligament cells and mouse periodontal tissue and its role in osteoblastic/cementoblastic differentiation.

A recent report showed that thymosin beta-4 (Tβ4) is expressed during the development of tooth germ, but its effect on osteoblastic/cementoblastic differentiation is a controversial topic. Furthermore, the precise expression and function of Tβ4 in periodontal tissue remains unclear. Therefore, the purpose of this study was to investigate the immunolocalization of Tβ4 in the developing periodontium of mouse, the function of Tβ4 in osteoblastic/cementoblastic differentiation, and the underlying mechanism regulating periodontal regeneration in human periodontal ligament cells (hPDLCs), cementoblasts, and osteoblasts. Tβ4 expression was observed in differentiating hPDLCs, osteoblasts of the periodontium during development, as well as in mature tissue. Higher Tβ4 expression was observed in hPDLCs than in cementoblasts and osteoblasts in the developing periodontium. The expression of Tβ4 mRNA and protein gradually increased during PDL cell differentiation. The downregulation of Tβ4 expression by Tβ4 siRNA transfection inhibited osteoblastic differentiation by decreasing calcium nodule formation, alkaline phosphatase (ALP) activity, and mRNA expression of differentiation markers in hPDLCs, cementoblasts, and osteoblasts. In contrast, Tβ4 activation using a Tβ4 peptide, promoted these processes by activation of Akt, p38, ERK MAPKs, and the NF-κB pathway. The expression of nuclear NFATc1 was upregulated by Tβ4 peptide in hPDLCs. Inhibition of the calcineurin/NFATc1 pathway by cyclosporin A and FK506, attenuated Tβ4-induced osteoblastic differentiation and activation of Wnt-related genes, as well as nuclear β-catenin in hPDLCs. In conclusion, this study demonstrates, for the first time, that Tβ4 is expressed in developing periodontal tissue and that its expression is associated with osteoblastic/cementoblastic differentiation. These results suggests that Tβ4 is a potential therapeutic target for periodontal regeneration or bone disease.

Thymosin Beta-4 Suppresses Osteoclastic Differentiation and Inflammatory Responses in Human Periodontal Ligament Cells

Background Recent reports suggest that thymosin beta-4 (Tβ4) is a key regulator for wound healing and anti-inflammation. However, the role of Tβ4 in osteoclast differentiation remains unclear. Purpose The purpose of this study was to evaluate Tβ4 expression in H2O2-stimulated human periodontal ligament cells (PDLCs), the effects of Tβ4 activation on inflammatory response in PDLCs and osteoclastic differentiation in mouse bone marrow-derived macrophages (BMMs), and identify the underlying mechanism. Methods Reverse transcription-polymerase chain reactions and Western blot analyses were used to measure mRNA and protein levels, respectively. Osteoclastic differentiation was assessed in mouse bone marrow-derived macrophages (BMMs) using conditioned medium (CM) from H2O2-treated PDLCs. Results Tβ4 was down-regulated in H2O2-exposed PDLCs in dose- and time-dependent manners. Tβ4 activation with a Tβ4 peptide attenuated the H2O2-induced production of NO and PGE2 and up-regulated iNOS, COX-2, and osteoclastogenic cytokines (TNF-α, IL-1β, IL-6, IL-8, and IL-17) as well as reversed the effect on RANKL and OPG in PDLCs. Tβ4 peptide inhibited the effects of H2O2 on the activation of ERK and JNK MAPK, and NF-κB in PDLCs. Furthermore, Tβ4 peptide inhibited osteoclast differentiation, osteoclast-specific gene expression, and p38, ERK, and JNK phosphorylation and NF-κB activation in RANKL-stimulated BMMs. In addition, H2O2 up-regulated Wnt5a and its cell surface receptors, Frizzled and Ror2 in PDLCs. Wnt5a inhibition by Wnt5a siRNA enhanced the effects of Tβ4 on H2O2-mediated induction of pro-inflammatory cytokines and osteoclastogenic cytokines as well as helping osteoclastic differentiation whereas Wnt5a activation by Wnt5a peptide reversed it. Conclusion In conclusion, this study demonstrated, for the first time, that Tβ4 was down-regulated in ROS-stimulated PDLCs as well as Tβ4 activation exhibited anti-inflammatory effects and anti-osteoclastogenesis in vitro. Thus, Tβ4 activation might be a therapeutic target for inflammatory osteolytic disease, such as periodontitis.

Thymosin beta-4 regulates activation of hepatic stellate cells via hedgehog signaling

The molecular mechanisms of thymosin beta-4 (TB4) involved in regulating hepatic stellate cell (HSC) functions remain unclear. Therefore, we hypothesize that TB4 influences HSC activation through hedgehog (Hh) pathway. HSC functions declined in a TB4 siRNA-treated LX-2. TB4 suppression down-regulated both integrin linked kinase (ILK), an activator of smoothened, and phosphorylated glycogen synthase kinase 3 beta (pGSK-3B), an inactive form of GSK-3B degrading glioblastoma 2 (GLI2), followed by the decreased expression of both smoothened and GLI2. A TB4 CRISPR also blocked the activation of primary HSCs, with decreased expression of smoothened, GLI2 and ILK compared with cells transfected with nontargeting control CRISPR. Double immunostaining and an immunoprecipitation assay revealed that TB4 interacted with either smoothened at the cytoplasm or GLI2 at the nucleus in LX-2. Smoothened suppression in primary HSCs using a Hh antagonist or adenovirus transduction decreased TB4 expression with the reduced activation of HSCs. Tb4-overexpressing transgenic mice treated with CCl4 were susceptible to the development hepatic fibrosis with higher levels of ILK, pGSK3b, and Hh activity, as compared with wild-type mice. These findings demonstrate that TB4 regulates HSC activation by influencing the activity of Smoothened and GLI2, suggesting TB4 as a novel therapeutic target in liver disease.

Angiogenic Induction by Trichinella spiralis Infection through Thymosin β4

Trichinella spiralis (T. spiralis) has been reported to induce angiogenesis and a supply of nutrients and to act as a reliable waste disposal system by induction of the expression of the angiogenic molecule vascular endothelial cell growth factor (VEGF) during nurse cell formation. However, the mechanism underlying the induction of VEGF in nurse cells by T. spiralis has not yet been defined. Some research has pointed to the possibility of hypoxia in nurse cells, but whether hypoxia occurs in infected muscle or nurse cells has not been studied. It is also a matter of debate whether hypoxia induces the expression of VEGF and subsequent angiogenesis in infected muscle. Recent studies showed that thymosin , a potent VEGF-inducing protein, was expressed at a very early stage of muscle infection by T. spiralis, suggesting that VEGF is induced at an early stage in nurse cells. Furthermore, hypoxia was not detected in any nurse cell stage but was detected in inflammatory cells. The findings suggest that induction of angiogenesis by VEGF in T. spiralis-infected nurse cells is mediated by thymosin and unrelated to hypoxia.

Interactions between Human Endogenous Retrovirus (HERV) and Human Immunodeficiency Virus (HIV)

Retroviruses genes have been inserted into the human genome for millions of years. These retroviruses are now inactive due to mutations such as deletions or nonsense mutations. After mutation, retroviruses eventually became fixed in the genome in their endogenous forms and existed as traces of ancient viruses. These retroviruses are called endogenous retroviruses (ERVs), with the human form known as human endogenous retrovirus. HERV cannot become a fully active virus, but a number of viral proteins or even virus particles are expressed under various conditions. Compared to endogenous retroviruses, some exogenous retroviruses are still infectious and can threaten human life. Among these, human immunodeficiency virus (HIV) is one of the most well-known and best-studied. Recent studies have shown some elements of HERV were activated by HIV infection and interact with HIV-derived proteins. In addition, many studies have attempted to use HERV as vaccination against HIV infection. This review will describe the regulation and interaction between HERV and HIV infection and mention the development of vaccines and therapeutic agents against HIV infection by using HERV elements.

Analysis of the Expression Patterns of Thymosin β4, Vascular Endothelial Growth Factor, and Hypoxia-Inducible Factor-1α in Various Tumors Using Tissue Microarray

Thymosin (TB-4) has been reported to play a key role in tumor growth, metastasis and angiogenesis. In addition, TB-4 induced the expression of vascular endothelial growth factor (VEGF) and stabilized the hypoxia-inducible factor (HIF)- in melanoma cells. Although the importance of thymosin in angiogenesis and metastasis has been proven, there are few studies that show the expression patterns of TB-4, VEGF and HIF-. This study was conducted to analyze the relationship among these proteins in various tumors. Using tissue microarray analysis, we investigated the expression patterns of TB-4, VEGF and HIF- in various tumors to identify the expression patterns and relationships of these proteins in certain tumors. TB-4 was highly expressed in osteosarcoma, colon adenocarcinoma, esophageal squamous cell carcinoma, kidney and urinary bladder transitional carcinoma, lung cancer, and liver cancer. HIF- was highly expressed in nasal cavity inverted papilloma, lung cancer, and esophageal squamous cell carcinoma. The expression patterns of TB-4 and HIF- were almost similar and co-localized. VEGF expression was high in the blood vessels in tumors, but usually not high in the tumors themselves. VEGF was moderately expressed in stomach cancer, liver angiosarcoma, gall bladder adenocarcinoma, and uterus endometrial adenocarcinoma. The expression patterns of VEGF shows similarities in certain tumors including stomach cancer, osteosarcoma, liposarcoma, lung cancer, liver cancer, gall bladder adenocarcinoma, esophageal squamous cell carcinoma, stomach cancer, colorectal carcinoma and renal cell carcinoma. These results suggest that the expression patterns of TB-4, HIF- and VEGF were co-localized and related to tumorigenesis and angiogenesis of certain tumors.