Fibroblasts in three dimensional matrices: cell migration and matrix remodeling

Sangmyung Rhee 생화학분자생물학회 2009 Experimental and molecular medicine Vol.41 No.12

Fibroblast-collagen matrix culture has facilitated the analysis of cell physiology under conditions that more closely resemble an in vivo-like environment compared to conventional 2-dimensional (2D) cell culture. Furthermore, it has led to significant progress in understanding reciprocal and adaptive interactions between fibroblasts and the collagen matrix, which occur in tissue. Recent studies on fibroblasts in 3-dimensional (3D) collagen matrices have revealed the importance of biomechanical conditions in addition to biochemical cues for cell signaling and migration. Depending on the surrounding mechanical conditions, cells utilize specific cytoskeletal proteins to adapt to their environment. More specifically, cells utilize microtubule dependent dendritic extensions to provide mechanical structure for matrix contraction under a low cell-matrix tension state, whereas cells in a high cell-matrix tension state utilize conventional acto-myosin activity for matrix remodeling. Results of collagen matrix contraction and cell migration in a 3D collagen matrix revealed that the use of appropriate growth factors led to promigratory and procontractile activity for cultured fibroblasts. Finally, the relationship between cell migration and tractional force for matrix remodeling was discussed. Fibroblast-collagen matrix culture has facilitated the analysis of cell physiology under conditions that more closely resemble an in vivo-like environment compared to conventional 2-dimensional (2D) cell culture. Furthermore, it has led to significant progress in understanding reciprocal and adaptive interactions between fibroblasts and the collagen matrix, which occur in tissue. Recent studies on fibroblasts in 3-dimensional (3D) collagen matrices have revealed the importance of biomechanical conditions in addition to biochemical cues for cell signaling and migration. Depending on the surrounding mechanical conditions, cells utilize specific cytoskeletal proteins to adapt to their environment. More specifically, cells utilize microtubule dependent dendritic extensions to provide mechanical structure for matrix contraction under a low cell-matrix tension state, whereas cells in a high cell-matrix tension state utilize conventional acto-myosin activity for matrix remodeling. Results of collagen matrix contraction and cell migration in a 3D collagen matrix revealed that the use of appropriate growth factors led to promigratory and procontractile activity for cultured fibroblasts. Finally, the relationship between cell migration and tractional force for matrix remodeling was discussed.

Cancer Microenvironment

Sangmyung Rhee 대한외과학회 2018 대한외과학회 학술대회 초록집 Vol.2018 No.11

Noonan syndrome-associated SHP2 mutation differentially modulates the expression of postsynaptic receptors according to developmental maturation

Oh, Jun-Young,Rhee, Sangmyung,Silva, Alcino J.,Lee, Yong-Seok,Kim, Hyong Kyu Elsevier 2017 Neuroscience Letters Vol.649 No.-

<P><B>Abstract</B></P> <P>Glutamate is the major excitatory neurotransmitter in the central nervous system, and related signaling involves both AMPA and NMDA subtype receptors. The expression of glutamate receptors is dynamically regulated during development. Recent studies showed that the dysregulation of glutamate receptor expression and function is associated with neurodevelopmental disorders including intellectual disability. Previously, a Noonan syndrome (NS)-associated SHP2 mutation (SHP2<SUP>D61G</SUP>) was shown to increase the synaptic delivery of AMPA receptor, subsequently impairing synaptic plasticity and learning in adult mice. However, how the mutant SHP2 affects glutamate receptor expression during development is not known. Here, we found that the SHP2<SUP>D61G</SUP> differentially regulates the expression of AMPA and NMDA receptors depending on the stage of neuronal maturation. In cultured neurons (immature stage; DIV 6), overexpression of SHP2<SUP>D61G</SUP> significantly increased the average size and the number of NMDA receptor-containing particles, but not those with AMPA receptors. In early matured neurons (DIV 12), SHP2<SUP>D61G</SUP> significantly increased only the average size of AMPA receptor particles, and subsequently increased their number in matured neurons (DIV 18). Importantly, all the changes described above for SHP2<SUP>D61G</SUP> neurons were reversed by inhibiting MAPK. These data demonstrate that the increased activation of MAPK signaling pathway by SHP2<SUP>D61G</SUP> could deregulate the surface expression of synaptic receptors during neuronal development, which likely contributes to cognitive impairments in NS patients.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A Noonan syndrome-associated mutation in SHP2 increases NMDA receptor expression in premature neurons. </LI> <LI> This mutation increases the size of AMPA receptor clusters in early maturing neurons. </LI> <LI> The SHP2 mutation also increases both the size and the number of AMPA receptor clusters in mature neurons. </LI> <LI> The altered expressions of glutamate receptors by mutant SHP2 can be reversed by inhibiting the MAPK signaling pathway. </LI> </UL> </P>

Epidermal growth factor improves the migration and contractility of aged fibroblasts cultured on 3D collagen matrices

KIM, DAEHWAN,KIM, SO YOUNG,MUN, SEOG KYUN,RHEE, SANGMYUNG,KIM, BEOM JOON UNKNOWN 2015 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE Vol.35 No.4

<P>Epidermal growth factor (EGF) plays a critical role in fibroblasts by stimulating the production of collagen and supports cell renewal through the interaction between keratinocytes and fibroblasts. It is well known that the contractile activity of fibroblasts is required for the remodeling of the extracellular matrix (ECM), which contributes to skin elasticity. However, the role of EGF in the contraction of aged fibroblasts under 3-dimensional (3D) culture conditions is not yet fully understood. In the present study, we demonstrated that young fibroblasts spread and proliferated more rapidly than aged fibroblasts under 2-dimensional (2D) culture conditions. Cell migration assay using a nested collagen matrix revealed that the migration of young fibroblasts was also greater than that of aged fibroblasts under 3D culture conditions. However, the addition of recombinant human EGF (rhEGF) resulted in the enhanced migration of aged fibroblasts; the migration rate was similar to that of the young fibroblasts. The aged fibroblasts showed decreased cluster formation compared with the young fibroblasts on the collagen matrix, which was improved by the addition of rhEGF. Furthermore, cell contraction assay revealed that the basal contractility of the aged fibroblasts was lower than that of the young fibroblasts; however, following treatment with rhEGF, the contractility was restored to levels similar or even higher to those of the young fibroblasts. Taken together, our results suggest that rhEGF is a potential renewal agent that acts to improve the migration and contraction of aged fibroblasts more efficiently than young fibroblasts under 3D culture conditions; thus, EGF may have valuable regenerative effects on aged skin.</P>

Histone demethylase KDM3B regulates the transcriptional network of cell-cycle genes in hepatocarcinoma HepG2 cells

An, Mi-Jin,Kim, Dae-Hyun,Kim, Chul-Hong,Kim, Mijin,Rhee, Sangmyung,Seo, Sang-Beom,Kim, Jung-Woong Elsevier 2019 Biochemical and biophysical research communication Vol.508 No.2

<P><B>Abstract</B></P> <P>Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most lethal cancer worldwide. Although gene mutations associated with HCC development have been intensively studied, how epigenetic factors specifically modulate the functional properties of HCC by regulating target gene expression is unclear. Here we demonstrated the overexpression of <I>KDM3B</I> in liver tissue of HCC patients using public RNA-seq data. Ablation of <I>KDM3B</I> by CRISPR/Cas9 retarded the cell cycle and proliferation of hepatocarcinoma HepG2 cells. Approximately 30% of <I>KDM3B</I> knockout cells exhibited mitotic spindle multipolarity as a chromosome instability (CIN) phenotype. RNA-seq analysis of <I>KDM3B</I> knockout revealed significantly down-regulated expression of cell cycle related genes, especially cell proliferation factor <I>CDC123</I>. Furthermore, the expression level of Cyclin D1 was reduced in <I>KDM3B</I> knockout by proteosomal degradation without any change in the expression of <I>CCND1</I>, which encodes Cyclin D1. The results implicate KDM3B as a crucial epigenetic factor in cell cycle regulation that manipulates chromatin dynamics and transcription in HCC, and identifies a potential gene therapy target for effective treatment of HCC.</P> <P><B>Highlights</B></P> <P> <UL> <LI> KDM3B overexpressed in human hepatocarcinoma cells. </LI> <LI> The ablation of <I>KDM3B</I> reduced proliferation of hepatocarcinoma HepG2 cells. </LI> <LI> Protein expression level of Cyclin D1 was down-regulated in <I>KDM3B</I> KO cells through the proteosomal degradation pathway. </LI> <LI> Transcript level of the cell proliferation factor <I>CDC123</I> was decreased in <I>KDM3B</I> KO cells. </LI> </UL> </P>

Spindle pole body component 25 homolog expressed by ECM stiffening is required for lung cancer cell proliferation

Jeong, Jangho,Keum, Seula,Kim, Daehwan,You, Eunae,Ko, Panseon,Lee, Jieun,Kim, Jaegu,Kim, Jung-Woong,Rhee, Sangmyung Elsevier 2018 Biochemical and biophysical research communication Vol.500 No.4

<P><B>Abstract</B></P> <P>Accumulating evidence has shown that matrix stiffening in cancer tissue by the deposition of extracellular matrix (ECM) is closely related with severe tumor progression. However, much less is known about the genes affected by matrix stiffness and its signaling for cancer progression. In the current research, we investigated the differential gene expression of a non-small lung adenocarcinoma cell line, H1299, cultured under the conditions of soft (∼0.5 kPa) and stiff (∼40 kPa) matrices, mimicking the mechanical environments of normal and cancerous tissues, respectively. For integrated transcriptome analysis, the genes identified by ECM stiffening were compared with 8248 genes retrieved from The Cancer Genome Atlas Lung Adenocarcinoma (TCGA). In stiff matrix, 29 genes were significantly upregulated, while 75 genes were downregulated. The screening of hazard ratios for these genes using the Kaplan-Meier Plotter identified 8 genes most closely associated with cancer progression under the condition of matrix stiffening. Among these genes, spindle pole body component 25 homolog (<I>SPC25</I>) was one of the most up-regulated genes in stiff matrix and tumor tissue. Knockdown of <I>SPC25</I> in H1299 cells using shRNA significantly inhibited cell proliferation with downregulation of the expression of checkpoint protein, Cyclin B1, under the condition of stiff matrix whereas the proliferation rate in soft matrix was not affected by <I>SPC25</I> silencing. Thus, our findings provide novel key molecules for studying the relationship of extracellular matrix stiffening and cancer progression.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Matrix stiffness regulates the expression of genes involved in cell proliferation. </LI> <LI> <I>SPC25</I> is identified as hazard gene induced by matrix stiffness. </LI> <LI> <I>SPC25</I> is involved in cancer cell proliferation under stiff matrix conditions. </LI> <LI> Depletion of <I>SPC25</I> induces G2/M arrest via chromosome misalignment in metaphase. </LI> </UL> </P>

Sox4-mediated caldesmon expression facilitates differentiation of skeletal myoblasts

Jang, Sang-Min,Kim, Jung-Woong,Kim, Daehwan,Kim, Chul-Hong,An, Joo-Hee,Choi, Kyung-Hee,Rhee, Sangmyung The Company of Biologists Limited 2013 Journal of cell science Vol.126 No.22

<P>Caldesmon (CaD), which was originally identified as an actin-regulatory protein, is involved in the regulation of diverse actin-related signaling processes, including cell migration and proliferation, in various cells. The cellular function of CaD has been studied primarily in the smooth muscle system; nothing is known about its function in skeletal muscle differentiation. In this study, we found that the expression of CaD gradually increased as differentiation of C2C12 myoblasts progressed. Silencing of CaD inhibited cell spreading and migration, resulting in a decrease in myoblast differentiation. Promoter analysis of the caldesmon gene (<I>Cald1</I>) and gel mobility shift assays identified Sox4 as a major trans-acting factor for the regulation of <I>Cald1</I> expression during myoblast differentiation. Silencing of <I>Sox4</I> decreased not only CaD protein synthesis but also myoblast fusion in C2C12 cells and myofibril formation in mouse embryonic muscle. Overexpression of CaD in <I>Sox4</I>-silenced C2C12 cells rescued the differentiation process. These results clearly demonstrate that CaD, regulated by <I>Sox4</I> transcriptional activity, contributes to skeletal muscle differentiation.</P>

Casein kinase 2 promotes the TGF-β-induced activation of α-tubulin acetyltransferase 1 in fibroblasts cultured on a soft matrix

( Eunae You ),( Jangho Jeong ),( Jieun Lee ),( Seula Keum ),( Ye Eun Hwang ),( Jee-hye Choi ),( Sangmyung Rhee ) 생화학분자생물학회 2022 BMB Reports Vol.55 No.4

Cell signals for growth factors depend on the mechanical properties of the extracellular matrix (ECM) surrounding the cells. Microtubule acetylation is involved in the transforming growth factor (TGF)-β-induced myofibroblast differentiation in the soft ECM. However, the mechanism of activation of α-tubulin acetyltransferase 1 (α-TAT1), a major α-tubulin acetyltransferase, in the soft ECM is not well defined. Here, we found that casein kinase 2 (CK2) is required for the TGF-β-induced activation of α-TAT1 that promotes microtubule acetylation in the soft matrix. Genetic mutation and pharmacological inhibition of CK2 catalytic activity specifically reduced microtubule acetylation in the cells cultured on a soft matrix rather than those cultured on a stiff matrix. Immunoprecipitation analysis showed that CK2α, a catalytic subunit of CK2, directly bound to the C-terminal domain of α-TAT1, and this interaction was more prominent in the cells cultured on the soft matrix. Moreover, the substitution of alanine with serine, the 236th amino acid located at the C-terminus, which contains the CK2-binding site of α-TAT1, significantly abrogated the TGF-β-induced microtubule acetylation in the soft matrix, indicating that the successful binding of CK2 and the C-terminus of α-TAT1 led to the phosphorylation of serine at the 236th position of amino acids in α-TAT1 and regulation of its catalytic activity. Taken together, our findings provide novel insights into the molecular mechanisms underlying the TGF-β-induced activation of α-TAT1 in a soft matrix. [BMB Reports 2022; 55(4): 192-197]

Role of the intracellular juxtamembrane domain of discoidin domain receptor 2 in focal adhesion formation

You, Eunae,Park, Soyeon,Kim, Daehwan,Jung, Jangho,Ko, Panseon,Park, Chan-Mi,Rhee, Sangmyung Taylor Francis 2014 Animal cells and systems Vol.18 No.6

Inhibition of Ku70 acetylation by INHAT subunit SET/TAF-Iβ regulates Ku70-mediated DNA damage response

Kim, Kee-Beom,Kim, Dong-Wook,Park, Jin Woo,Jeon, Young-Joo,Kim, Daehwan,Rhee, Sangmyung,Chae, Jung-Il,Seo, Sang-Beom Springer-Verlag 2014 Cellular and molecular life sciences Vol.71 No.14