A hemidesmosomal protein regulates actin dynamics and traction forces in motile keratinocytes

Hiroyasu, Sho; Colburn, Zachary; Jones, Jonathan

doi:10.1096/fj.201500160r

Cited by 30 publications

(34 citation statements)

References 51 publications

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“…Conversely, collagen XVII expression is increased at the leading edge during wound healing [38] and at the invasive front of carcinomas [15], suggesting that it promotes motility. Consistently, COL17A1 supports directed migration by stabilizing actin bundles, which generates traction forces [37]. Together, this suggests that cancer cells may increase the invasive potential by either up- or downregulating collagen XVII expression.…”

Section: Discussionmentioning

confidence: 79%

“…This thesis is supported by several previous observations. In keratinocytes, complete loss of Col17a1 expression increases cell motility [36] and partial reduction of COL17A1 levels promotes undirected motility [37]. Conversely, collagen XVII expression is increased at the leading edge during wound healing [38] and at the invasive front of carcinomas [15], suggesting that it promotes motility.…”

Section: Discussionmentioning

confidence: 99%

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In epithelial cancers, aberrant COL17A1 promoter methylation predicts its misexpression and increased invasion

et al. 2016

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BackgroundMetastasis is a leading cause of death among cancer patients. In the tumor microenvironment, altered levels of extracellular matrix proteins, such as collagens, can facilitate the first steps of cancer cell metastasis, including invasion into surrounding tissue and intravasation into the blood stream. However, the degree of misexpression of collagen genes in tumors remains understudied, even though this knowledge could greatly facilitate the development of cancer treatment options aimed at preventing metastasis.MethodsWe systematically evaluate the expression of all 44 collagen genes in breast cancer and assess whether their misexpression provides clinical prognostic significance. We use immunohistochemistry on 150 ductal breast cancers and 361 cervical cancers and study DNA methylation in various epithelial cancers.ResultsIn breast cancer, various tests show that COL4A1 and COL4A2 overexpression and COL17A1 (BP180, BPAG2) underexpression provide independent prognostic strength (HR = 1.25, 95% CI = 1.17–1.34, p = 3.03 × 10−10; HR = 1.18, 95% CI = 1.11–1.25, p = 8.11 × 10−10; HR = 0.86, 95% CI = 0.81–0.92, p = 4.57 × 10−6; respectively). Immunohistochemistry on ductal breast cancers confirmed that the COL17A1 protein product, collagen XVII, is underexpressed. This strongly correlates with advanced stage, increased invasion, and postmenopausal status. In contrast, immunohistochemistry on cervical tumors showed that collagen XVII is overexpressed in cervical cancer and this is associated with increased local dissemination. Interestingly, consistent with the opposed direction of misexpression in these cancers, the COL17A1 promoter is hypermethylated in breast cancer and hypomethylated in cervical cancer. We also find that the COL17A1 promoter is hypomethylated in head and neck squamous cell carcinoma, lung squamous cell carcinoma, and lung adenocarcinoma, in all of which collagen XVII overexpression has previously been shown.ConclusionsParadoxically, collagen XVII is underexpressed in breast cancer and overexpressed in cervical and other epithelial cancers. However, the COL17A1 promoter methylation status accurately predicts both the direction of misexpression and the increased invasive nature for five out of five epithelial cancers. This implies that aberrant epigenetic control is a key driver of COL17A1 gene misexpression and tumor cell invasion. These findings have significant clinical implications, suggesting that the COL17A1 promoter methylation status can be used to predict patient outcome. Moreover, epigenetic targeting of COL17A1 could represent a novel strategy to prevent metastasis in patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0290-6) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 79%

Section: Discussionmentioning

confidence: 99%

In epithelial cancers, aberrant COL17A1 promoter methylation predicts its misexpression and increased invasion

et al. 2016

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“…Traction force microscopy was performed as described elsewhere (Hiroyasu et al, ). Briefly, fibroblasts were plated onto coverslips coated with polyacrylamide gel containing Alexa‐647 fluorescent beads.…”

Section: Methodsmentioning

confidence: 99%

Nesprin‐2G knockout fibroblasts exhibit reduced migration, changes in focal adhesion composition, and reduced ability to generate traction forces

Woychek

Jones

2019

Cytoskeleton

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The nuclear envelope protein nesprin‐2G is a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex and is responsible for mechanical and signaling crosstalk between the nucleus and cytoskeleton. A prior study has demonstrated that nesprin‐2G knockout mice show delayed wound healing. The goal was to elucidate the mechanism underlying the delayed wound closure in this mouse model. Primary fibroblasts from wild‐type and knockout neonatal mice were isolated. Knockout cells exhibited decreased focal adhesion (FA) size, number, and intensity. Consistent with this result, FA protein expression levels were decreased in knockout cells. Additionally, knockout fibroblasts displayed an abnormal actin cytoskeleton, as evidenced by loss of TAN line formation and both cytoplasmic and peri‐nuclear actin staining. Using collective and single cell motility assays, it was found that knockout cells exhibited a reduction in both speed and directed migration. Traction force microscopy revealed that knockout fibroblasts generated fewer traction forces compared with WT fibroblasts. In summary, the data indicated that changes in actin organization and defects in FAs result in a reduced ability of knockout fibroblasts to generate traction forces needed for efficient motility.

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“…The impact of β-PIX KD on FAs, cytoskeleton organization and MLC activation led us to investigate whether these changes result in modulation of the traction forces exerted by cells on their substrate. To do so, we plated HaCaT cells onto a fluorescent bead-embedded gel substrate as described previously (Eisenberg et al, 2013;Hiroyasu et al, 2016). We then compared traction force localization and magnitudes exerted by a control cells, β-PIX KD HaCaT clones and GFP-tagged β-PIX expressing β-PIX KD HaCaT cells.…”

Section: Traction Force Magnitude Is Increased In β-Pix Kd Hacat Cellsmentioning

confidence: 99%

“…Traction force microscopy was performed as described elsewhere (Eisenberg et al, 2013;Hiroyasu et al, 2016). In brief, cells were plated on polyacrylamide gels embedded with fluorescent beads.…”

Section: Traction Force Microscopymentioning

confidence: 99%

Loss of β-PIX inhibits focal adhesion disassembly and promotes keratinocyte motility via myosin light chain activation

Hiroyasu

Stimac

Hopkinson

et al. 2017

Journal of Cell Science

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During healing of the skin, the cytoskeleton of keratinocytes and their matrix adhesions, including focal adhesions (FAs), undergo reorganization. These changes are coordinated by small GTPases and their regulators, including the guanine nucleotide exchange factor β-PIX (also known as ARHGEF7). In fibroblasts, β-PIX activates small GTPases, thereby enhancing migration. In keratinocytes in vitro, β-PIX localizes to FAs. To study β-PIX functions, we generated β-PIX knockdown keratinocytes. During wound closure of β-PIX knockdown cell monolayers, disassembly of FAs is impaired, and their number and size are increased. In addition, in the β-PIX knockdown cells, phosphorylated myosin light chain (MLC; also known as MYL2) is present not only in the leading edge of cells at the wound front, but also in the cells following the front, while p21-activated kinase 2 (PAK2), a regulator of MLC kinase (MYLK), is mislocalized. Inhibition or depletion of MYLK restores FA distribution in β-PIX knockdown cells. Traction forces generated by β-PIX knockdown cells are increased relative to those in control cells, a result consistent with an unexpected enhancement in the migration of single β-PIX knockdown cells and monolayers of such cells. We propose that targeting β-PIX might be a means of promoting epithelialization of wounds in vivo.

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A hemidesmosomal protein regulates actin dynamics and traction forces in motile keratinocytes

Cited by 30 publications

References 51 publications

In epithelial cancers, aberrant COL17A1 promoter methylation predicts its misexpression and increased invasion

In epithelial cancers, aberrant COL17A1 promoter methylation predicts its misexpression and increased invasion

Nesprin‐2G knockout fibroblasts exhibit reduced migration, changes in focal adhesion composition, and reduced ability to generate traction forces

Loss of β-PIX inhibits focal adhesion disassembly and promotes keratinocyte motility via myosin light chain activation

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