Scratch Wound Repair Research Articles

The TLK1-MK5 Axis Regulates Motility, Invasion, and Metastasis of Prostate Cancer Cells.

Background: Metastatic dissemination of prostate cancer (PCa) accounts for the majority of PCa-related deaths. However, the exact mechanism of PCa cell spread is still unknown. We uncovered a novel interaction between two unrelated promotility factors, tousled-like kinase 1 (TLK1) and MAPK-activated protein kinase 5 (MK5), that initiates a signaling cascade promoting metastasis. In PCa, TLK1−MK5 signaling might be crucial, as androgen deprivation therapy (ADT) leads to increased expression of both TLK1 and MK5 in metastatic patients, but in this work, we directly investigated the motility, invasive, and metastatic capacity of PCa cells following impairment of the TLK1 > MK5 axis. Results: We conducted scratch wound repair and transwell invasion assays with LNCaP and PC3 cells to determine if TLK1 and MK5 can regulate motility and invasion. Both genetic depletion and pharmacologic inhibition of TLK1 and MK5 resulted in reduced migration and invasion through a Matrigel plug. We further elucidated the potential mechanisms underlying these effects and found that this is likely due to the reorganization of the actin fibers at lamellipodia and the focal adhesions network, in conjunction with increased expression of some MMPs that can affect penetration through the ECM. PC3, a highly metastatic cell line when assayed in xenografts, was further tested in a tail-vein injection/lung metastasis model, and we showed that, following inoculation, treatment with GLPG0259 (MK5 specific inhibitor) or J54 (TLK1 inhibitor) resulted in the lung tumor nodules being greatly diminished in number, and for J54, also in size. Conclusion: Our data support that the TLK1−MK5 axis is functionally involved in driving PCa cell metastasis and clinical aggressiveness; hence, disruption of this axis may inhibit the metastatic capacity of PCa.

Abstract 2424: TLK1-MK5 axis modulates actin filaments and focal adhesion components to promote PCa cell migration and invasion

Abstract Background: Metastatic dissemination of prostate cancer (PCa) accounts for majority of PCa related deaths. However, the exact mechanism of PCa cell spread is still unknown. We uncovered a novel interaction between two unrelated promotility factors, tousled-like kinase 1 (TLK1) and MAPK-activated protein kinase 5 (MK5), which initiates a signaling cascade promoting metastasis. In PCa, TLK1-MK5 signaling might be crucial as androgen deprivation therapy (ADT) leads to increased expression of both TLK1 and MK5 in metastatic patients. Moreover, TCGA analysis also revealed higher expression of both TLK1 and MK5 in metastatic tumors. Therefore, we hypothesize that TLK1-MK5 axis promotes PCa cell migration and invasion through actin reorganization as well as focal adhesion proteins modification and disruption of this interaction may help to keep the tumor localized. Methods and Results: We conducted scratch wound repair and proliferation assays to determine if TLK1 and MK5 can regulate motility and proliferation. Both genetic depletion and pharmacologic inhibition of TLK1 and MK5 resulted in reduced migration of both non-malignant MEF and PCa cells without affecting cellular proliferation rate. Trans-well invasion assays also demonstrated reduced invasion of MK5−/− MEF cells in Matrigel and other ECMs. However, TLK1 overexpression alone in the MK5−/− MEF cells did not increase the wound healing and invasion which suggested that TLK1 cannot enhance cellular migration and invasion in absence of MK5. We confirmed TLK1 binding of MK5 by co-IP, His- and GST-pull down assays and established that TLK1 phosphorylates MK5 on three residues (S160, S354, S386) that results in the catalytic activation of MK5. While WB detected substantial amount of pMK5 S354 and TLK1 in all major PCa cell lines, anti-androgen treatment increases pMK5 S354 level in a dose-dependent manner and pharmacologic inhibition of TLK1 reduces pMK5 S354 level in LNCaP cells, which suggest TLK1 as an authentic pMK5 S354 kinase. IHC analysis of TRAMP mice and human PCa TMA also revealed higher pMK5 S354 level in metastatic lesions compared to the low-risk tumors. In addition, pMK5 S354 was found to colocalize with neuroendocrine marker chromogranin A, together of which may suggest pMK5 as a potential biomarker of PCa aggressiveness. The motility reduction of PCa cells upon MK5 inhibition is a consequence of impaired actin filamentation as we observed in LNCaP and PC3 cells. Our WB analysis demonstrated that MK5 inhibition by a repurposed inhibitor results in decreased FAK Y861, Paxillin Y118, HSP27 S78/82 phosphorylation, and ERK3 stabilization, which could lead to altered actin filamentation, focal adhesions, morphology, as well as motility and invasion. Conclusion: Our data support that TLK1-MK5 axis is involved in driving PCa cell metastasis and clinical aggressiveness, hence, disruption of this axis may inhibit the metastasis of PCa. Citation Format: Md Imtiaz Khalil, Vibha Singh, Judy A. King, Arrigo De Benedetti. TLK1-MK5 axis modulates actin filaments and focal adhesion components to promote PCa cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2424.

TLK1‐MK5 Axis Modulates Actin Filaments and Focal Adhesion Components to Promote PCa Cell Migration and Invasion

BackgroundMetastatic dissemination of prostate cancer (PCa) accounts for majority of PCa related deaths. However, the exact mechanism of PCa cell spread is still unknown. We uncovered a novel interaction between two unrelated promotility factors, tousled‐like kinase 1 (TLK1) and MAPK‐activated protein kinase 5 (MK5), which initiates a signaling cascade promoting metastasis. In PCa, TLK1‐MK5 signaling might be crucial as androgen deprivation therapy (ADT) leads to increased expression of both TLK1 and MK5 in metastatic patients. Moreover, TCGA analysis also revealed higher expression of both TLK1 and MK5 in metastatic tumors. Therefore, we hypothesize that TLK1‐MK5 axis promotes PCa cell migration and invasion through actin reorganization as well as focal adhesion proteins modification and disruption of this interaction may help to keep the tumor localized.Methods and ResultsWe conducted scratch wound repair and proliferation assays to determine if TLK1 and MK5 can regulate motility and proliferation. Both genetic depletion and pharmacologic inhibition of TLK1 and MK5 resulted in reduced migration of both non‐malignant MEF and PCa cells without affecting cellular proliferation rate. Trans‐well invasion assays also demonstrated reduced invasion of MK5−/− MEF cells in Matrigel and other ECMs. However, TLK1 overexpression alone in the MK5−/− MEF cells did not increase the wound healing and invasion which suggested that TLK1 cannot enhance cellular migration and invasion in absence of MK5. We confirmed TLK1 binding of MK5 by co‐IP, His‐ and GST‐pull down assays and established that TLK1 phosphorylates MK5 on three residues (S160, S354, S386) that results in the catalytic activation of MK5. While western blotting (WB) detected substantial amount of pMK5 S354 and TLK1 in all major PCa cell lines, anti‐androgen treatment increases pMK5 S354 level in a dose‐dependent manner and pharmacologic inhibition of TLK1 reduces pMK5 S354 level in LNCaP cells, which suggest TLK1 as an authentic pMK5 S354 kinase. IHC analysis of TRAMP mice and human PCa TMA also revealed higher pMK5 S354 level in metastatic lesions compared to the low‐risk tumors. In addition, pMK5 S354 was found to colocalize with neuroendocrine marker chromogranin A, together of which may suggest pMK5 as a potential biomarker of PCa aggressiveness. The motility reduction of PCa cells upon MK5 inhibition is a consequence of impaired actin filamentation as we observed in LNCaP and PC3 cells. Our WB analysis demonstrated that MK5 inhibition by a repurposed inhibitor results in decreased FAK Y861, Paxillin Y118, HSP27 S78/82 phosphorylation, and ERK3 stabilization, which could lead to altered actin filamentation, focal adhesions, morphology, as well as motility and invasion.ConclusionOur data support that TLK1‐MK5 axis is functionally involved in driving PCa cell metastasis and clinical aggressiveness, hence, disruption of this axis may inhibit the metastatic capacity of PCa.

Effects of silencing neuropilin-2 on proliferation, migration, and invasion of colorectal cancer HT-29

ABSTRACT To investigate the effects of silencing neuropilin-2(NRP-2) on the proliferation, migration, and invasion of colorectal cancer(CRC) HT-29. Lipofectamine 2000 was used to transfect specific siRNA for NRP-2 and nonspecific control siRNA into human colorectal cancer HT-29 as the transfection group and meaningless sequence group. HT-29 cultured in a medium was used as the blank control group. The expression levels of NRP-2 mRNA in the cells were detected by real-time fluorescence quantitative PCR. The expressions of proliferation-associated protein Ki-67 in the cells were detected by immunochemical staining. Migration ability was assessed by a monolayer cell scratch wound damage and repair experiment. The Transwell chamber invasion experiment was adopted to determine invasive ability by measuring the number of tumor cells crossing the chamber membrane. Compared with the meaningless sequence group and blank control group, real-time fluorescence quantitative PCR showed that the relative expression level of NRP-2 mRNA in the transfection group was significantly decreased(P < 0.05). Results of immunochemical staining revealed that the expression of Ki-67 protein in the transfected cells was significantly reduced, and the proliferation ability was decreased(P < 0.05). The results further showed that the scratch healing rate of the transfected cells decreased after 24 h of healing(P < 0.05). Results of Transwell invasion assay showed that the number of cells passing through the stromal membrane of the upper chamber to the back of the chamber was significantly reduced in the transfection group(p < 0.05). Silencing NRP-2 could inhibit the proliferation, migration, and invasion of colorectal cancer HT-29.

Upregulation of IGSF3 expression by iron chelation leads to the inhibition of epithelial cell proliferation

Background/Significance We recently demonstrated that the Immunoglobulin Superfamily-3 (IGSF3), a transmembrane tetraspanin interacting protein, is required for scratch wound repair, cell migration, and barrier function, suggesting a potential key role in lung injury repair. However, the role of IGSF3 in epithelial cell proliferation remain unknown. Furthermore, it is unclear how IGSF3 expression is affected in acute respiratory distress syndrome (ARDS), characterized by hypoxia, loss of lung epithelial barrier, and insufficient cell repair. We hypothesized that hypoxia alters IGSF3 expression and that IGSF3 is mechanistically involved in the effects of hypoxia on human bronchial epithelial cell proliferation. Methods To mimic hypoxia, we exposed human bronchial epithelial cells (Beas2B) submerged in cell culture media to iron chelator deferoxamine (DFO, 100uM, 16-24hr). Proliferation was measured using CCK8 assay. We stably knocked down IGSF3 in Beasw2B using CRISPR-CAS9. Tetraspanin enriched microdomains (TEMs) were isolated using density gradient ultracentrifugation. Single cell RNA-seq (scRNA-seq) was performed on deidentified human ARDS (n=1) and non-diseased (n=3) lungs. Results DFO caused a significant decrease in epithelial cell proliferation (by~ 25%, p<0.001) concomitant to a markedly increased IGSF3 gene and protein expression (by 2- and 2.5-fold, respectively, p<0.01) that was prevented by treatment with the HIF-1a inhibitor, KCF72 (p<0.01). IGSF3-deficient Beas2B exhibited increased cell proliferation at baseline (by~20-50% vs control cells, 24hr; p<0.01) and following treatment with DFO (by ~ 200% vs DFO-treated control cells; 24hr, p<0.01). Compared to non-diseased lungs, scRNA-seq of ARDS lungs revealed marked enrichment of IGSF3 in basal epithelial cell. Interestingly, a similar pattern was observed in Tspan5, an IGSF3 binding tetraspanin induced by iron chelation and in CD147, a hypoxia-inducible Immuhnoglobulin Superfamily protein (Basigin, EMMPRIN). We confirmed that IGSF3 co-localized with Tspan5 within TEMs in Beas2B and, interestingly found that both proteins co-localized with CD147. Conclusions IGSF3 may be induced by hypoxia in lung epithelial cells such as basal cells where it may inhibit cell proliferation and therefore repair. Interestingly, loss of IGSF3 has also a detrimental effect on processes involved in repair such as cell migration and barrier function. This suggests that IGSF3 homeostasis is important in epithelial cell function, possibly through interactions with membrane complexes such as tetraspanins and other transmembrane proteins such as the matrix-regulating CD147.

Nanodiamonds inhibit scratch-wound repair in lung epithelial cell monolayers by blocking cell migration and inhibiting cell proliferation

Proliferation and migration of lung epithelial cells following the injury to the epithelial lining of alveoli and airways in the lung are pivotal for remodeling and repair of the wound to restore normal lung function. In the present study, we examined the modulatory effect of carboxylated nanodiamonds (cNDs) on the cell division, migration, and adhesion of epithelial cells in the well-established in vitro model of wound repair and cell migration. Flow cytometry and confocal microscopy results indicated that both LA4 and A549 cells effectively internalized fluorescent carboxylated nanodiamonds (cFNDs) and the internalized nanodiamonds were essentially localized in the cytoplasmic region. Treatment with cNDs blocked the division and migration of cells to fill the scratch wound. Live cell imaging and time-lapse videography of the wound healing process indicated a significant inhibition of cell proliferation activity in cND-treated cells and blocked the wound repair process. Trans-well cell-migration assay results further support the inhibitory effect of cNDs on the cell migration process. Western blotting and immunofluorescence staining indicated that the crucial proteins involved in epithelial-mesenchymal transition (EMT) and cell migration i.e. β-catenin, Vimentin, NM-myosin, and Focal Adhesion Kinase (FAK) were downregulated after treatment with cNDs, while the expression of E-cadherin and Claudin-1, major cell adhesion markers remained unaltered. Taken together, our results indicate that the decline in cell proliferation activity, downregulation in the expression of various crucial protein like β-Catenin, NM-myosin, FAK, and Vimentin involved in the cell migration and unaltered expression of cell adhesion molecules E-cadherin and Claudin-1, may be the factors that contribute to the cND-mediated inhibition of EMT during the wound repair process in the monolayers of lung epithelial cells.

Abstract 4906: TLK1-MK5 signaling axis contributes to prostate cancer cell motility and invasion

Abstract Background: Prostate cancer (PCa) is the 2nd leading death-causing malignancy in men where the majority of fatalities occur due to metastasis of cancer cells to adjacent and distal organs. We identified the novel interaction between two kinases that may initiate a signaling cascade promoting metastasis. One kinase is MAPK activated protein kinase 5 (MAPKAPK5 or MK5) which plays a debated role in cellular motility along with other cellular processes. Our observations are that MK5 interacts with another S/T kinase, called tousled like kinase 1 (TLK1), which may also have a potential role in cellular motility which is largely unstudied, apart from one report in breast cancer on TLK2. In PCa, TLK1-MK5 signaling axis might be crucial as androgen deprivation therapy leads to increased expression of both TLK1 and MK5 in metastatic castrate-resistant prostate cancer patients. Our working hypothesis is that TLK1-MK5 axis promotes PCa cell motility and invasion through actin rearrangement as well as focal adhesion proteins modification and disruption of this interaction may help to keep the tumor localized. Method and Results: A previous proteomic screening from our lab identified MK5 as one of the novel interactors of TLK1 with high confidence (p&amp;lt;0.00005). To validate this interaction, we overexpressed EGFP-MK5 in HEK 293 cells and performed a reciprocal Co-IP study targeting both TLK1 and EGFP-MK5 that confirmed their interaction in vivo. Additionally, a reciprocal His- and GST-pull down assays demonstrated the direct and physical interaction between TLK1 and MK5 in vitro. Another Co-IP experiment revealed that TLK1-MK5 interaction starts with the attachment of the cells to the ECM. We conducted several scratch wound repair and 3D chemotactic migration assays to find out if TLK1-MK5 axis enhances the migration rate of the cells. Inhibition of the kinase activity of TLK1 by using a specific inhibitor (thioridazine) decreases the migration rate of the HEK 293 cells, which supports the importance of TLK1 function in motility. Wound repair and chemotactic migration were also demonstrated to be faster in overexpressed EGFP-MK5 in LNCaP and MEF cells and compared also to MK5 knock out cells. An in-vitro kinase assay using [y-32P] ATP demonstrated that incubation of purified TLK1B and purified MK5 increases the phosphorylation of MK5 and its kinase activity. Mass spectrometric analysis identified three unique phosphorylation sites in MK5 (S160, S354, S386) by TLK1B which are mapped to the functional domains of MK5. We also measured the protein level of several focal adhesion proteins in TLK1 and MK5 co-expressed HEK 293 cells such as total FAK, phospho-FAK (Tyr397), total Paxillin, Phospho-Paxillin (Tyr118, Ser178), etc. and observed activating phosphorylation. Conclusion: Our data support that TLK1-MK5 interaction is functionally involved in cell motility and migration and hence, disruption of this axis may inhibit the metastasis of PCa. Citation Format: Md Imtiaz Khalil, Vibha Singh, Arrigo De Benedetti. TLK1-MK5 signaling axis contributes to prostate cancer cell motility and invasion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4906.

Inorganic polyphosphate in platelet rich plasma accelerates re-epithelialization invitro and invivo.

Wound healing requires well-coordinated events including hemostasis, inflammation, proliferation, and remodeling. Delays in any of these stages leads to chronic wounds, infections, and hypertrophic scarring. Burn wounds are particularly problematic, and may require intervention to ensure timely progression to reduce morbidity and mortality. To accelerate burn wound healing, Platelet-Rich Plasma (PRP)1 can be of value, since platelets release growth factor proteins and inorganic polyphosphates (polyP) that may be integral to wound healing. We used polyP-depleted keratinocyte (HaCaT) and fibroblast cell culture models to determine cell proliferation and scratch-wound repair to determine if polyP, platelet lysate, or combined treatment could accelerate wound healing. While polyP and PRP significantly reduced the open scratch-wound area in fibroblasts and keratinocytes, polyP had no effect on keratinocyte or fibroblast proliferation. PRP was also evaluated as a treatment in a murine model of full thickness wound healing in vivo, including a treatment in which PRP was supplemented with purified polyP. PRP induced significantly more rapid re-epithelialization by Day 3. Pure polyP enhanced the effects of PRP on epithelial tongues, which were significantly elongated in the PRP + high-dose polyP treatment groups compared to PRP alone. Thus, PRP and polyP may serve as an effective therapeutic combination for treating wounds.

HS2ST1-dependent signaling pathways determine breast cancer cell viability, matrix interactions, and invasive behavior.

Heparan sulfate proteoglycans (HSPGs) act as signaling co‐receptors by interaction of their sulfated glycosaminoglycan chains with numerous signaling molecules. In breast cancer, the function of heparan sulfate 2‐O‐sulfotransferase (HS2ST1), the enzyme mediating 2‐O‐sulfation of HS, is largely unknown. Hence, a comparative study on the functional consequences of HS2ST1 overexpression and siRNA knockdown was performed in the breast cancer cell lines MCF‐7 and MDA‐MB‐231. HS2ST1 overexpression inhibited Matrigel invasion, while its knockdown reversed the phenotype. Likewise, cell motility and adhesion to fibronectin and laminin were affected by altered HS2ST1 expression. Phosphokinase array screening revealed a general decrease in signaling via multiple pathways. Fluorescent ligand binding studies revealed altered binding of fibroblast growth factor 2 (FGF‐2) to HS2ST1‐expressing cells compared with control cells. HS2ST1‐overexpressing cells showed reduced MAPK signaling responses to FGF‐2, and altered expression of epidermal growth factor receptor (EGFR), E‐cadherin, Wnt‐7a, and Tcf4. The increased viability of HS2ST1‐depleted cells was reduced to control levels by pharmacological MAPK pathway inhibition. Moreover, MAPK inhibitors generated a phenocopy of the HS2ST1‐dependent delay in scratch wound repair. In conclusion, HS2ST1 modulation of breast cancer cell invasiveness is a compound effect of altered E‐cadherin and EGFR expression, leading to altered signaling via MAPK and additional pathways.

Ginsenoside Rg1-induced activation of astrocytes promotes functional recovery via the PI3K/Akt signaling pathway following spinal cord injury

AimsTo determine whether ginsenoside Rg1 is involved in scratch wound healing through altered expression of related molecules in astrocytes and improved functional recovery after spinal cord injury (SCI). Materials and methodsAstrocytes were isolated from rats, followed by Rg1 treatment. The wound healing test was performed to observe the scratch wound healing in different groups. The expression of nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), and components of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway were detected by western blot. Reverse transcription–polymerase chain reaction (RT-PCR) was used to measure the altered expression of laminin (LN) and fibronectin (FN). A revised Allen's method for the SCI model was performed, followed by Rg1 treatment. Then, functional scoring was conducted to evaluate the functional recovery. Hematoxylin-eosin (HE) staining showed changes in the void area. Finally, western blot assessed the expression of glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs). Key findingsRg1 mediated scratch wound healing through inducing an increased release of LN, FN, NGF, GDNF, and bFGF in vitro. Additionally, Rg1 activated the PI3K/Akt signaling pathway and promoted the functional recovery of hindlimb movement in rats. Furthermore, Rg1 significantly reduced the void area and downregulated the expression of GFAP and CSPGs. SignificanceRg1 not only enhanced the scratch wound repair in vitro through the release of astroglial neurotrophic factors, adhesion factors, and inhibitory factors, but it also improved the functional recovery in vivo following SCI.

Vitamin D attenuates lung injury via stimulating epithelial repair, reducing epithelial cell apoptosis and inhibits TGF-β induced epithelial to mesenchymal transition

Vitamin D regulates cell proliferation, inhibits cytokines release at sites of inflammation and reduces inflammatory responses. In this study, the aim was to investigate whether exogenous vitamin D attenuates LPS-induced lung injury via modulating epithelial cell proliferation, migration, apoptosis and epithelial mesenchymal transition (EMT). Murine and in vitro primary type II alveolar epithelial cell work were included in this study. In vivo, mice were mildly vitamin D deficient, 0.1, 1.5, 10 mg/kg 1,25(OH)2-vitamin D3 or 25(OH)-vitamin D3 was administrated by means of an intra-gastric injection for 14 days pre-intra-tracheal (IT) LPS, which remarkedly promoted alveolar epithelial type II cells proliferation, inhibited ATII cells apoptosis and inhibited EMT, with the outcome of attenuated LPS-induced lung injury. In vitro, vitamin D stimulated epithelial cell scratch wound repair, reduced primary ATII cells apoptosis as well. Vitamin D promoted primary human ATII cells proliferation through the PI3K/AKT signaling pathway and activation of vitamin D receptor (VDR). Moreover, vitamin D inhibited EMT in response to TGF-β, which was vitamin D receptor dependent. In conclusion, vitamin D attenuates lung injury via stimulating ATII cells proliferation and migration, reducing epithelial cell apoptosis and inhibits TGF-β induced EMT. Together, these results suggest that vitamin D has therapeutic potential for the resolution of ARDS.

The Implications of TLK1‐MK5 Signaling Axis in Prostate Cancer Cell Motility and Invasion

BackgroundProstate cancer (PCa) is the 2nd leading death‐causing malignancy in men where the majority of fatalities occur due to metastasis of cancer cells to adjacent and distal organs. Devising a therapeutic intervention requires the identification of underlying signaling pathways involved in the progression of PCa. We identified the novel interaction between two kinases that may initiate a signaling cascade promoting metastasis. One kinase is MAPK activated protein kinase 5 (MAPKAPK5 or MK5) which plays a debated role in cellular motility along with other cellular processes. Our observations are that MK5 interacts with another S/T kinase, called tousled like kinase 1 (TLK1), which may also have a potential role in cellular motility which is largely unstudied, apart from one report in breast cancer on TLK2. In PCa, TLK1‐MK5 signaling axis might be crucial as androgen deprivation therapy leads to increased expression of both TLK1 and MK5 in metastatic castrate‐resistant prostate cancer patients. Our working hypothesis is that TLK1‐MK5 axis promotes PCa cell motility and invasion through actin rearrangement as well as focal adhesion proteins modification and disruption of this interaction may help to keep the tumor localized.Method and ResultsA previous protoarray interaction study from our lab identified MK5 as one of the novel interactors of TLK1 with high confidence (p&lt;0.00005). To validate this interaction, we overexpressed EGFP‐MK5 in HEK 293 cells and performed a reciprocal Co‐IP study targeting both TLK1 and EGFP‐MK5 that confirmed their interaction in vivo. Additionally, a reciprocal His‐ and GST‐pull down assays demonstrated the direct and physical interaction between TLK1 and MK5 in vitro. Another Co‐IP experiment revealed that TLK1‐MK5 interaction starts with the attachment of the cells to the ECM. We conducted several scratch wound repair assays to find out if TLK1‐MK5 axis enhances the migration rate of the cells. Inhibition of the kinase activity of TLK1 by using a specific inhibitor (thioridazine) decreases the migration rate of the HEK 293 cells, which supports the importance of TLK1 function in motility. Wound repair was also demonstrated to be faster in overexpressed EGFP‐MK5 in LNCaP and MEF cells and compared also to MK5 knock out cells. An in‐vitro Kinase assay using [y‐32P] ATP demonstrated that incubation of purified TLK1B and purified MK5 increases the phosphorylation of MK5, but not on TLK1B. Another in‐vitro kinase assay using HSP27 as a substrate of MK5 revealed that TLK1B increases the kinase activity of MK5 towards HSP27 phosphorylation. Mass spectrometric analysis identified three unique phosphorylation sites in MK5 (S160, S354, S386) by TLK1B which are mapped to the kinase, NLS/NES and ERK3 binding domain of MK5. We also measured the protein level of several focal adhesion proteins in TLK1 and MK5 co‐expressed HEK 293 cells such as total FAK, phospho‐FAK (Tyr397), total Paxillin, Phospho‐Paxillin (Tyr118, Ser178), etc. and observed activating phosphorylation.ConclusionOur data support that TLK1‐MK5 interaction is functionally involved in cell motility and migration and hence, disruption of this axis may inhibit the metastasis of PCa.Support or Funding InformationDoD‐PCRP grant W81XWH‐17‐1‐0417

Model Systems to Investigate NOX-Dependent Cell Migration and Invasiveness.

There is mounting evidence indicating that reactive oxygen species (ROS) play a crucial role in cell migration and invasion. Our previous studies have demonstrated the NADPH oxidase (NOX) family of enzymes are a source of ROS in different cell types undergoing migration. Several NOX enzymes are induced or activated in processes including wound repair and maintenance of epithelial barriers, as well as in promoting metastatic cell migration and invasiveness. This chapter outlines three different in vitro assays used to examine how NOX enzymes are involved in cell motility: scratch-wound repair, Matrigel invasion, and migration from confluent cell monolayer boundaries created by cell culture inserts. The three methods provide a range of experimental approaches for delineating roles of NOX enzymes in cell migration through manipulation of the expression or activities of the endogenous or overexpressed oxidases.

123 - Nox4 through mutant p53 induces a pro-inflammatory and pro-migratory secretome in the tumor microenvironment

The tumor microenvironment (TME) is a complex environment of tumor cells and resident stromal cells that are surrounded by secreted factors and extracellular matrix (ECM) proteins. Recent studies suggestreactive oxygen species (ROS) plays an important role in tumor progression and stromal cell activation.Previously, we showed wild-type (WT) and mutant (mt) forms of p53 differentially regulate ROS generation by NADPH oxidase-4 (NOX4). We found that WT-p53 suppresses TGF-β-induced NOX4, ROS production, and cell migration, whereas tumor-associated mt-p53 proteins enhance NOX4 expression and cell migration by TGF-β/SMAD3-dependent mechanisms. In this study, we investigated the role of mutant p53-induced NOX4 on the cancer cell secretome and the effect NOX4-derived ROS has on the TME. We found that conditioned media collected from H1299 lung epithelial cells stably expressing either mutant p53-R248Q or R273H promotes the migration and invasion of naïve H1299 cells in scratch-wound repair and trans-well invasion assays, respectively. Furthermore, conditioned media from cells co-transfected with mutant p53 and dominant negative NOX4 (Nox4-DN) elicited diminished migratory behavior by naïve H1299 cells. We utilized a Western blot-based cytokine array analysis to identify inflammatory factors in the H1299 mutant p53 CM that promote cell migration and invasion. We found CCL5 was significantly reduced in CM from H1299 cells expressing p53-R248Q and NOX4-DN. Collectively, our findings provide further insight into NOX4-based regulation of communication between cells in the tumor microenvironment and its potential as a therapeutic target affecting inflammation-driven metastatic disease.

Defibrotide Stimulates Angiogenesis and Protects Endothelial Cells from Calcineurin Inhibitor-Induced Apoptosis via Upregulation of AKT/Bcl-xL.

Sinusoidal obstruction syndrome is a life-threatening complication that can occur after haematopoietic stem cell transplantation. Defibrotide (DF) has been approved for the treatment of individuals with severe sinusoidal obstruction syndrome following haematopoietic stem cell transplantation in the European Union and the United States. However, the precise mechanisms by which DF protects endothelial cells remain to be elucidated. In this study, we found that DF stimulated angiogenesis in vitro and in vivo as assessed by vascular tube formation, scratch-wound repair and Matrigel plug assays. These effects were associated with an activation of pro-survival signalling pathways, including AKT (protein kinase B), ERK (extracellular signal-regulated kinases) and p38. More importantly, DF alleviated calcineurin inhibitor-induced growth inhibition and apoptosis of human umbilical vein endothelial cells and human hepatic sinusoidal endothelial cells in parallel with upregulation of anti-apoptotic protein B-cell lymphoma-extra-large (Bcl-xL), which was mediated by AKT (protein kinase B). Notably, these effects were abrogated when Bcl-xL was depleted by small interfering RNA (ribonucleic acid). In addition, DF counteracted calcineurin inhibitor-induced activation of nuclear factor-κB and Janus kinase 2 (JAK2)/Signal Transducer and Activator of Transcription 3 (STAT3) signalling and production of cytokines in vascular endothelial cell-derived EA.hy926 cells. Taken together, DF has pro-angiogenic, anti-apoptotic and anti-inflammatory effects on endothelial cells. DF is a potentially useful agent to prevent the development of, and treat individuals with, endothelial cell injury-related complications after haematopoietic stem cell transplantation.

Expression of stomach cancer-associated transcript 16 in gastric cancer tissues and its effects on gastric cancer biological behavior

Objective To study the expression of long non-coding RNA (LncRNA) stomach cancer-associated transcript 16 (STCAT16) in gastric cancer tissues and its effects on the proliferation, migration and invasion of gastric cancer cells. Methods The different expression of STCAT16 in 32 cases of gastric carcinoma and corresponding adjacent tissues was detected by real-time fluorescence quantitative polymerase chain reaction (PCR). The STCAT16 overexpression plasmid and empty vector was separately transfected gastric cancer cell line AGS with low expression of STCAT16. The cell proliferation of empty vector group, non-transfection group and STCAT16 analogue transfection group was evaluated by cell counting kit-8 (CCK-8) assay at 0, 24, 48, 72 and 96 hours after transfection. The colony forming ability was tested by colony formation assay. The cell invasion ability was measured by Transwell chamber assay and migration ability was tested by scratch-wound assay. The effects of STCAT16 on tumorigenicity in vivo were verified by tumorigenicity experiments in nude mice. T-test and one-way analysis of variance were performed for data analysis. Repeated measures analysis of variance was used to compare the repeated measured data among groups. Chi square test and Fisher exact probability method were used for comparison of counting data. Results The expression of STCAT16 in gastric cancer tissues was low (0.87±0.19), while it was high in corresponding adjacent tissues (2.32±0.37), and the difference was statistically significant (t=-20.859, P<0.05). The expression of STCAT16 of STCAT16 analogue transfection group was higher than that of empty vector group (3.43±0.25 vs 1.00±0.06), and the difference was statistically significant (t=-16.795, P<0.05). Compared to empty vector group and non-transfection group, the cell proliferation decreased in STCAT16 analogue transfection group at 72 and 96 hours after transfection (1.41±0.07, 1.42±0.08, 1.03±0.09, and 1.72±0.11, 1.78±0.14, 1.24±0.08, respectively), and the differences were statistically significant (t=15.043, 5.358, 12.193 and 8.109, all P<0.05). The results of colony formation assay indicated that the colony forming ability of gastric cells in STCAT16 analogue transfection group was lower than that in empty vector group (97.3±9.1 vs 185.0±20.1) and non-transfection group (97.3±9.1 vs 138.0±11.1), and the differences were statistically significant (t=11.634 and 4.417, both P<0.05). The results of Transwell assay showed that the number of AGS cells passing through the membrane of STCAT16 analogue transfection group was significantly less than those of empty vector group and non-transfection group (151.0±28.1 vs 228.0±38.2 and 151.0±28.1 vs 199.3±17.9), and the differences were statistically significant (t=4.823 and 4.747, both P<0.05). After transfection for 48 hours and 72 hours, the scratch-wound repair rate of STCAT16 analogue transfection group decreased, compared with those of empty vector group and non-transfection group ((52.67±6.11)%, (53.33±5.51)%, (42.67±4.72)%, and (90.67±2.51)%, (90.60±5.41)%, (69.67±1.52)%, respectively), and the differences were statistically significant (t=5.773, 5.955, 21.000 and 5.881, all P<0.05). The results of tumorigenicity in nude mice showed that compared with those of empty vector group, the tumor size of STCAT16 analogue transfection group was smaller at one-, two-, three- and four-week ((0.42±0.10) cm3 vs (0.16±0.05) cm3, (0.66±0.13) cm3 vs (0.34±0.05) cm3, (1.25±0.22) cm3 vs (0.54±0.13) cm3, (2.54±0.46) cm3 vs (0.78±0.41) cm3)), and the differences were statistically significant (t=3.175, 4.190, 7.996 and 9.705, all P<0.05). Conclusions STCAT16 is lowly expressed in gastric cancer tissues. The proliferation, migration, invasion ability and tumorigenicity in nude mice of gastric cancer cell is inhibited after upregulating the expression of STCAT16. Key words: Stomach neoplasms; STCAT16; Long non-coding RNA; Cell proliferation; Cell movement

Effects of MMP12 on cell motility and inflammation during corneal epithelial repair

Corneal epithelial defects are a common cause of ocular morbidity and can result in corneal scarring if they do not heal properly. Matrix metalloproteinases (MMPs) are extracellular matrix proteinases that regulate multiple aspects of corneal repair. We have previously shown that MMP12 has a protective effect on corneal fibrosis through its regulation of neutrophil and macrophage infiltration and angiogenesis in a chemical injury model involving full thickness damage to the cornea. However, the role of MMP12 in injuries limited to the corneal epithelium is relatively unknown. This study investigates the reparative effects of MMP12 following isolated corneal epithelial injury. Using a corneal epithelial debridement injury model performed on corneas of wild-type (WT) mice, we show that Mmp12 is expressed early following corneal epithelial injury with highest expression levels at 8 h after injury and lower expression levels at 4 and 8 days after injury. We investigated whether MMP12 has an effect on the rate of epithelial repair and cell migration using in vivo and in vitro scratch assays performed on WT and Mmp12-/- mice. We found that loss of MMP12 results in a slower scratch wound repair rate both in vivo and in vitro. We also found that corneas of Mmp12-/- mice have decreased neutrophil infiltration following injury. Loss of MMP12, however, does not affect cell proliferation in the center of the wounds. These data support a role of MMP12 in promoting early repair processes following corneal epithelial injury by enhancing epithelial cell migration and neutrophil infiltration.

Exogenous H2S modulates mitochondrial fusion-fission to inhibit vascular smooth muscle cell proliferation in a hyperglycemic state.

AimVascular smooth muscle cell (VSMC) proliferation in response to hyperglycemia is an important process in the development of arterial vessel hyperplasia. The shape change of mitochondria is dynamic and closely related to fission and fusion. Hydrogen sulfide (H2S) was confirmed to have anti-oxidative, anti-inflammatory and anti-proliferative effects. However, little it is known about its effects on mitochondrial morphology induced by hyperglycemia. The aim of the study is to demonstrate that H2S inhibits VSMC proliferation through regulating mitochondrial fission.Methods and resultsWe observe lower H2S levels as well as higher proliferative protein expression levels for proliferative cell nuclear antigen (PCNA) and cyclin D1 and higher mitochondrial fusion–fission protein expression levels for dynamin-related protein 1 (Drp 1) in human kidney arteries and in db/db mouse aorta. Exogenous H2S (100 μM NaHS) inhibits vascular smooth muscle cells of human pulmonary aorta(HPASMC) proliferation and migration in response to high glucose using the BrdU and scratch wound repair assays, decreases proliferative protein (PCNA and cyclin D1) expression, and reduces ROS production in the cytoplasm and mitochondria. When HPASMCs proliferate with a high glucose treatment, the mitochondria become small spheres with a short rod-shaped structure, whereas NaHS, a mitochondrial division inhibitor and siDrp prevent VSMC proliferation and maintain mitochondria as stationary and randomly dispersed with fixed structures.ConclusionExogenous H2S aids in inhibiting mitochondrial fragmentation and affects proliferation in db/db mice and HPASMCs by decreasing Drp 1 expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-016-0102-x) contains supplementary material, which is available to authorized users.

Hepatocyte Growth Factor Gene-Modified Mesenchymal Stem Cells Augment Sinonasal Wound Healing.

This study was designed to investigate the effects of hepatocyte growth factor (HGF) transgenic mesenchymal stem cells (HGF-MSCs) on wound healing in the sinonasal mucosa and nasal epithelial cells (NECs). We also sought to determine whether HGF-MSCs and MSCs can migrate into the injured mucosa and differentiate into ciliated cells. Human HGF-overexpressing umbilical cord MSCs (hHGF-UCMSCs) were established, and upregulation of hHGF expression was confirmed by real-time PCR (RT-PCR) and enzyme-linked immunosorbant assay (ELISA). To investigate the paracrine effect of human MSCs (hMSCs) on nasal epithelial repair, hMSC- and HGF-MSC-conditioned media (CM) were used in NEC proliferation assays and in an in vitro scratch-wound repair model. The in vivo sinonasal wound-healing model was established, and all enrolled rabbits were randomly assigned to four groups: the GFP-MSC group, the HGF-MSC group, the Ad-HGF group, and the surgery control group. The average decreased diameter was recorded, and the medial wall of the maxillary sinus was removed for histological analysis and scanning electron microscopy. Collagen deposition in the wound tissue was detected via Masson trichrome (M&T) staining. The distribution of MSCs and HGF-MSCs was observed by immunofluorescence. MSCs improved nasal wound healing both in vivo and in vitro. HGF overexpression in MSCs augmented the curative effects. Reduced collagen deposition and transforming growth factor beta1 (TGF-β1) expression were detected in the HGF-MSC group compared with the MSC-, Ad-HGF-, and phosphate-buffered saline-treated groups based on M&T staining and ELISA. The enhanced therapeutic effects of HGF-MSCs were accompanied by decreased level of the fibrogenic cytokine TGF-β1. In addition, both HGF-MSCs and MSCs can migrate to the injured mucosa and epithelial layer.

HIF1α Dependent CXCR4/SDF1 Signaling Promotes Alveolar Type II Cell Spreading and the Restitution of Epithelial Barrier Integrity After Lung Injury

Rationale. Alveolar type (AT) II cell spreading is a critical mechanism for the restitution of epithelial barrier integrity after ATI cell sloughing during lung injury. We hypothesized that Hypoxia Inducible Factor (HIF) 1α signaling promotes ATII cell spreading and the restoration of barrier function. Results. HIF was activated in ATII cells in ODD-Luc (HIF reporter) mice treated with intratracheal (i.t.) LPS, keratinocyte chemokine (KC), or hydrochloric acid (HCl), as shown by costaining for proSPC and luciferase or glucose transporter (GLUT) 1. To elucidate whether HIF1α promotes barrier restoration via cell spreading, we treated mice with i.t. KC, which causes ATI cell sloughing followed by rapid resolution of epithelial permeability associated with ATII cell spreading but independent of proliferation. The resolution of epithelial permeability was delayed in Sftpc-rtTA;tetOCre;HIF1αf/f mice but accelerated by HIF stabilization with i.p. dimethyloxalylglycine (DMOG). In cultured MLE-12 and/or primary rat ATII cells, DMOG accelerated whereas HIF1α shRNA delayed repair of a scratch wound, an assay of cell spreading. A microarray on rat ATII cells after adenoviral overexpression of HIF1α revealed upregulation of both CXCR4 and its ligand SDF1. CXCR4 and SDF1 were upregulated in ATII cells in lung injury in vivo. Finally, scratch wound repair was accelerated by rSDF1 and delayed by the CXCR4 inhibitor AMD3100. Conclusions HIF1α promotes ATII cell spreading and the restitution of epithelial integrity after lung injury. The reparative role of HIF1α may be mediated by autocrine CXCR4/SDF1 signaling.