PRP4 Induces Epithelial-Mesenchymal Transition and Drug Resistance in Colon Cancer Cells via Activation of p53.

Pre-mRNA processing factor 4B (PRP4) promotes pre-mRNA splicing and signal transduction. Recent studies have shown that PRP4 modulates the assembly of actin cytoskeleton in cancer cells and induces epithelial-mesenchymal transition (EMT) and drug resistance. PRP4 displays kinase domain-like cyclin-dependent kinases and mitogen-activated protein kinases, making it capable of phosphorylating p53 and other target proteins. In the current study, we report that PRP4 induces drug resistance and EMT via direct binding to the p53 protein, inducing its phosphorylation. Moreover, PRP4 overexpression activates the transcription of miR-210 in a hypoxia-inducible factor 1α (HIF-1α)-dependent manner, which activates p53. The involvement of miR-210 in the activation of p53 was confirmed by utilizing si-miR210. si-miR210 blocked the PRP4-activated cell survival pathways and reversed the PRP4-induced EMT phenotype. Moreover, we used deferoxamine as a hypoxia-mimetic agent, and si-HIF to silence HIF-1α. This procedure demonstrated that PRP4-induced EMT and drug resistance emerged in response to consecutive activation of HIF-1α, miR-210, and p53 by PRP4 overexpression. Collectively, our findings suggest that the PRP4 contributes to EMT and drug resistance induction via direct interactions with p53 and actions that promote upregulation of HIF-1α and miR-210. We conclude that PRP4 is an essential factor promoting cancer development and progression. Specific PRP4 inhibition could benefit patients with colon cancer.

An Overview About the Role of Adaptive Immunity in Keeping SARS-CoV-2 Reinfections at Bay.

Coronavirus disease 2019 (COVID-19) is a worldwide emergency that has affected millions of populations in developed and underdeveloped countries. To our surprise, many people have been tested positive twice. Few cases of true reinfections involved genetic alterations in the virus. Appearance of multiple positive tests may be due to human errors or remnant genetic material, but genetic modification in virus represents very serious issue of controlling this pandemic. It is the need of the day that all the gaps and deficiencies, represented by variable response of adaptive immune system toward this infection, be filled and rectified. We have discussed reinfections with variable outcomes along with the possible reasons for variable response. Phenomena such as T cell memory, absence of cross-reactive immunity, T cell exhaustion, drawbacks pertaining to neutralizing antibodies, and immune enhancement are crucial areas by which adaptive immune response can weaken considerably. Earlier and stronger herd immunity is also at the mercy of strong adaptive immune system to avoid future pandemics by the same microorganism. Likewise, consequences of this phenomenon should also be considered during vaccine development as resources worth billions are being used and staked. Many countries have entered the second/third waves of COVID-19. Therefore, we need to come up with ways toward uniform strengthening of adaptive immune response to fight off this pandemic. Also, to develop and maintain constant resistance to severe acute respiratory syndrome coronavirus (SARS-CoV-2), the mentioned weakened links in the chain of adaptive immunity may be explored to keep viral invasion and physiological damage to minimum.

Potential applications of bacterial cellulose and its composites for cancer treatment.

Bacterial cellulose (BC) has received immense interest in medical, pharmaceutical, and other related fields owing to its intrinsic physical, mechanical, and biological features. Its structural features offer an ideal environment for developing composites, thereby further extending its areas of applications. BC was initially used in wound dressing, artificial blood vessels, organ development, and tissue regeneration; however, the recent focus has switched to 3D printing techniques. BC can serve as suitable material for treating different cancers due to unique liquid absorbing and drug loading properties. BC-based scaffolds have been synthesized and tested for in vitro culturing of cancer cells to simulate tumor microenvironments. These scaffolds support normal growth of cancer cells, particularly breast and ovarian cancer cells, showing significant adhesion, proliferation, ingrowth, and differentiation. This review describes the different approaches of manipulating BC for use in medicine, with particular focus on the applications of BC composites in cancer treatment. A detailed discussion about various formulations of BC in multiple cancer therapeutics is summarized.

An Update on the Role of Dietary Phytochemicals in Human Skin Cancer: New Insights into Molecular Mechanisms.

Human skin is continuously subjected to environmental stresses, as well as extrinsic and intrinsic noxious agents. Although skin adopts various molecular mechanisms to maintain homeostasis, excessive and repeated stresses can overwhelm these systems, leading to serious cutaneous damage, including both melanoma and non-melanoma skin cancers. Phytochemicals present in the diet possess the desirable effects of protecting the skin from damaging free radicals as well as other benefits. Dietary phytochemicals appear to be effective in preventing skin cancer and are inexpensive, widely available, and well tolerated. Multiple in vitro and in vivo studies have demonstrated the significant anti-inflammatory, antioxidant, and anti-angiogenic characteristics of dietary phytochemicals against skin malignancy. Moreover, dietary phytochemicals affect multiple important cellular processes including cell cycle, angiogenesis, and metastasis to control skin cancer progression. Herein, we discuss the advantages of key dietary phytochemicals in whole fruits and vegetables, their bioavailability, and underlying molecular mechanisms for preventing skin cancer. Current challenges and future prospects for research are also reviewed. To date, most of the chemoprevention investigations have been conducted preclinically, and additional clinical trials are required to conform and validate the preclinical results in humans.

Significance of Green Synthetic Chemistry from a Pharmaceutical Perspective.

BACKGROUND: Conventional practices of synthesis, manufacturing, and processing have led to severe adverse consequences for living beings and the environment. OBJECTIVES: Although medications cannot be replaced, the methods of synthesizing, manufacturing, and processing them can be changed and/or replaced. This paper explains the significance of green chemistry practices in the pharmaceutical industry. It emphasizes that we must replace conventional drug synthesis, processing, and manufacturing techniques with greener ones that are cost-effective, sustainable, environment-friendly, and profitable. DISCUSSION: This paper comprises five sections. Section 1 is an introduction to green chemistry and its correlation with the pharmaceutical industry. Section 2 discusses the metrics necessary to measure the greenness of a process. Section 3 is about solvents used in the pharmaceutical industry, hazards, safety status, and environmental effects, including the ozone depletion potential. Section 4 explains catalytic amidation reactions because amides are one of the most commonly occurring functional groups with pharmacological activity. Section 5 discusses successful cases of converting conventional synthesis of active pharmaceutical ingredients and/or their intermediates to greener, sustainable alternatives. CONCLUSION: A balance is necessary between profits, processes, consumers, and the environment to ensure the survival of all stakeholders and decrease the environmental burden of pharmaceuticals. Incentives such as green chemistry awards should be endorsed and encouraged, in addition to making green chemistry part of tertiary education. In addition, changes to rules and regulations for drug approval in the context of green chemistry principles are necessary in order to preserve our planet for future generations.

PRP4 Kinase Domain Loss Nullifies Drug Resistance and Epithelial-Mesenchymal Transition in Human Colorectal Carcinoma Cells.

We have investigated the involvement of the pre-mRNA processing factor 4B (PRP4) kinase domain in mediating drug resistance. HCT116 cells were treated with curcumin, and apoptosis was assessed based on flow cytometry and the generation of reactive oxygen species (ROS). Cells were then transfected with PRP4 or pre-mRNA-processing-splicing factor 8 (PRP8), and drug resistance was analyzed both in vitro and in vivo. Furthermore, we deleted the kinase domain in PRP4 using GatewayTM technology. Curcumin induced cell death through the production of ROS and decreased the activation of survival signals, but PRP4 overexpression reversed the curcumin-induced oxidative stress and apoptosis. PRP8 failed to reverse the curcumin-induced apoptosis in the HCT116 colon cancer cell line. In xenograft mouse model experiments, curcumin effectively reduced tumour size whereas PRP4 conferred resistance to curcumin, which was evident from increasing tumour size, while PRP8 failed to regulate the curcumin action. PRP4 overexpression altered the morphology, rearranged the actin cytoskeleton, triggered epithelial-mesenchymal transition (EMT), and decreased the invasiveness of HCT116 cells. The loss of E-cadherin, a hallmark of EMT, was observed in HCT116 cells overexpressing PRP4. Moreover, we observed that the EMT-inducing potential of PRP4 was aborted after the deletion of its kinase domain. Collectively, our investigations suggest that the PRP4 kinase domain is responsible for promoting drug resistance to curcumin by inducing EMT. Further evaluation of PRP4-induced inhibition of cell death and PRP4 kinase domain interactions with various other proteins might lead to the development of novel approaches for overcoming drug resistance in patients with colon cancer.

Ionizing Radiation Regulates Vascular Endothelial Growth Factor-A Transcription in Cultured Human Vascular Endothelial Cells Via the PERK/eIF2α/ATF4 Pathway.

PURPOSE: The delivery of high-dose hypofractionated radiation to a tumor induces vascular damage, but little is known about the responses of vascular endothelial cells to high-dose radiation. We examined whether high-dose irradiation alters vascular endothelial growth factor (VEGF) signaling, which is a critical regulator of the functional integrity and viability of vascular endothelial cells. METHODS AND MATERIALS: Human umbilical vein endothelial cells and human coronary artery endothelial cells were treated with 5, 10, 20, or 30 Gy ionizing radiation (IR). Expression values of VEGFA mRNA were analyzed by real-time polymerase chain reaction at 4 hours after irradiation and normalized to the average value of mock-irradiated human umbilical vein endothelial cell or human coronary artery endothelial cell controls. RESULTS: Irradiation with doses higher than 10 Gy causes an acute increase in VEGFA transcript levels, which was accompanied by activation of the PERK/eIF2α/activating transcription factor 4 (ATF4) pathway in human vascular endothelial cells. ATF4 knockdown with siRNA completely prevented the IR-induced upregulation of VEGFA transcripts, and chromatin immunoprecipitation assays demonstrated that ATF4 binding to the VEGFA locus was enriched in response to IR. Postirradiation treatment with an intracellular inhibitor of VEGF signaling significantly enhances high-dose IR-induced apoptosis in human vascular endothelial cells. CONCLUSIONS: Human vascular endothelial cells activate PERK/eIF2α/ATF4/VEGF signaling in response to high-dose IR to mitigate the apoptotic response. Thus, for cancer treatment, intracellular inhibitors of VEGF signaling could be employed to enhance stereotactic body radiation therapy-induced vascular damage, which would augment tumor cell death.

Dopamine receptor D2 activation suppresses the radiosensitizing effect of aripiprazole via activation of AMPK.

Drug repositioning has garnered attention as an alternative strategy to the discovery and development of novel anticancer drug candidates. In this study, we screened 321 FDA-approved drugs against nonirradiated and irradiated MCF-7 cells, revealing that aripiprazole, a dopamine receptor D2 (D2R) partial agonist, enhances the radiosensitivity of MCF-7 cells. Unexpectedly, D2R-selective antagonist treatment significantly enhanced the radiosensitizing effects of aripiprazole and prevented aripiprazole-induced 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. Direct AMPK activation with A769662 treatment blunted the radiosensitizing effects of aripiprazole. These results indicate that aripiprazole has potential as a radiosensitizing drug. Furthermore, prevention of D2R/AMPK activation might enhance these anticancer effects of aripiprazole in breast cancer cells.

Auranofin, an Anti-rheumatic Gold Drug, Aggravates the Radiation-Induced Acute Intestinal Injury in Mice.

Pelvic and abdominal radiotherapy plays an important role in eradication of malignant cells; however, it also results in slight intestinal injury. The apoptosis of cells in the intestinal epithelium is a primary pathological factor that initiates radiation-induced intestinal injury. Auranofin, a gold-containing triethylphosphine, was approved for the treatment of rheumatoid arthritis, and its therapeutic application has been expanded to a number of other diseases, such as parasitic infections, neurodegenerative disorders, AIDS, and bacterial infections. Recently, a treatment strategy combining the use of auranofin and ionizing radiation aimed at increasing the radiosensitivity of cancer cells was proposed for improving the control of local cancers. In this study, we evaluated the effect of auranofin on the radiosensitivity of intestinal epithelial cells. The treatment with a combination of 1 µM auranofin and 5 Gy ionizing radiation showed clear additive effects on caspase 3 cleavage and apoptotic DNA fragmentation in IEC-6 cells, and auranofin administration significantly aggravated the radiation-induced intestinal injury in mice. Auranofin treatment also resulted in the activation of the unfolded protein response and in the inhibition of thioredoxin reductase, which is a key component of the cellular antioxidant system. Pre-treatment with N-acetyl cysteine, a well-known scavenger of reactive oxygen species, but not with a chemical chaperone, which inhibits endoplasmic reticulum stress and the ensuing unfolded protein response, significantly reduced the radiosensitizing effects of auranofin in the IEC-6 cells. In addition, transfection of IEC-6 cells with a small interfering RNA targeted against thioredoxin reductase significantly enhanced the radiosensitivity of these cells. These results suggest that auranofin-induced radiosensitization of intestinal epithelial cells is mediated through oxidative stress caused by the deregulation of thioredoxin redox system, and auranofin treatment can be an independent risk factor for the development of acute pelvic radiation disease.

CRISPR/Cas9-mediated genome editing of splicing mutation causing congenital hearing loss.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has ushered in a new era of gene therapy. In this study, we aimed to demonstrate precise CRISPR/Cas9-mediated genome editing of the splicing mutation c.919-2A > G in intron 7 of the SLC26A4 gene, which is the second most common causative gene of congenital hearing loss. We designed candidate single-guide RNAs (sgRNAs) aimed to direct the targeting of Staphylococcus aureus Cas9 to either exon 7 or exon 8 of SLC26A4. Several of the designed sgRNAs showed targeting activity, with average indel efficiencies ranging from approximately 14% to 25%. The usage of dual sgRNAs delivered both into Neuro2a cells and primary mouse embryonic fibroblasts resulted in the successful removal of large genomic fragments within the target locus. We subsequently evaluated genome editing in the presence of artificial donor templates to induce precise target modification via homology-directed repair. Using this approach, two different donor plasmids successfully introduced silent mutations within the c.919-2A region of Slc26a4 without evident off-target activities. Overall, these results indicate that CRISPR/Cas9-mediated correction of mutations in the Slc26a4 gene is a feasible therapeutic option for restoration of hearing loss.

PRP4 kinase induces actin rearrangement and epithelial-mesenchymal transition through modulation of the actin-binding protein cofilin.

Cell actin cytoskeleton is primarily modulated by Rho family proteins. RhoA regulates several downstream targets, including Rho-associated protein kinase (ROCK), LIM-Kinase (LIMK), and cofilin. Pre-mRNA processing factor 4B (PRP4) modulates the actin cytoskeleton of cancer cells via RhoA activity inhibition. In this study, we discovered that PRP4 over-expression in HCT116 colon cancer cells induces cofilin dephosphorylation by inhibiting the Rho-ROCK-LIMK-cofilin pathway. Two-dimensional gel electrophoresis, and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) analysis indicated increased expression of protein phosphatase 1A (PP1A) in PRP4-transfected HCT116 cells. The presence of PRP4 increased the expression of PP1A both at the mRNA and protein levels, which possibly activated cofilin through dephosphorylation and subsequently modulated the cell actin cytoskeleton. Furthermore, we found that PRP4 over-expression did not induce cofilin dephosphorylation in the presence of okadaic acid, a potent phosphatase inhibitor. Moreover, we discovered that PRP4 over-expression in HCT116 cells induced dephosphorylation of migration and invasion inhibitory protein (MIIP), and down-regulation of E-cadherin protein levels, which were further restored by the presence of okadaic acid. These findings indicate a possible molecular mechanism of PRP4-induced actin cytoskeleton remodeling and epithelial-mesenchymal transition, and make PRP4 an important target in colon cancer.

PRPF overexpression induces drug resistance through actin cytoskeleton rearrangement and epithelial-mesenchymal transition.

Pre-mRNA processing factor (PRPF) 4B kinase belongs to the CDK-like kinase family, and is involved in pre-mRNA splicing, and in signal transduction. In this study, we observed that PRPF overexpression decreased the intracellular levels of reactive oxygen species, and inhibited resveratrol-induced apoptosis by activating the cell survival signaling proteins NFκB, ERK, and c-MYC in HCT116 human colon cancer cells. PRPF overexpression altered cellular morphology, and rearranged the actin cytoskeleton, by regulating the activity of Rho family proteins. Moreover, it decreased the activity of RhoA, but increased the expression of Rac1. In addition, PRPF triggered the epithelial-mesenchymal transition (EMT), and decreased the invasiveness of HCT116, PC3 human prostate, and B16-F10 melanoma cells. The loss of E-cadherin, a hallmark of EMT, was observed in HCT116 cells overexpressing PRPF. Taken together, these results indicate that PRPF blocks the apoptotic effects of resveratrol by activating cell survival signaling pathways, rearranging the actin cytoskeleton, and inducing EMT. The elucidation of the mechanisms that underlie anticancer drug resistance and the anti-apoptosis effect of PRPF may provide a therapeutic basis for inhibiting tumor growth and preventing metastasis in various cancers.

Rac1 promotes chondrogenesis by regulating STAT3 signaling pathway.

The small GTPase protein Rac1 is involved in a wide range of biological processes including cell differentiation. Previously, Rac1 was shown to promote chondrogenesis in micromass cultures of limb mesenchyme. However, the pathways mediating Rac1's role in chondrogenesis are not fully understood. This study aimed to explore the molecular mechanisms by which Rac1 regulates chondrogenic differentiation. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) was increased as chondrogenesis proceeded in micromass cultures of chick wing bud mesenchyme. Inhibition of Rac1 with NSC23766, janus kinase 2 (JAK2) with AG490, or STAT3 with stattic inhibited chondrogenesis and reduced phosphorylation of STAT3. Conversely, overexpression of constitutively active Rac1 (Rac L61) increased phosphorylation of STAT3. Rac L61 expression resulted in increased expression of interleukin 6 (IL-6), and treatment with IL-6 increased phosphorylation of STAT3. NSC23766, AG490, and stattic prohibited cell aggregation, whereas expression of Rac L61 increased cell aggregation, which was reduced by stattic treatment. Our studies indicate that Rac1 induces STAT3 activation through expression and action of IL-6. Overexpression of Rac L61 increased expression of bone morphogenic protein 4 (BMP4). BMP4 promoted chondrogenesis, which was inhibited by K02288, an activin receptor-like kinase-2 inhibitor, and increased phosphorylation of p38 MAP kinase. Overexpression of Rac L61 also increased phosphorylation of p38 MAPK, which was reduced by K02288. These results suggest that Rac1 activates STAT3 by expression of IL-6, which in turn increases expression and activity of BMP4, leading to the promotion of chondrogenesis.

Staurosporine induces chondrogenesis of chick embryo wing bud mesenchyme in monolayer cultures through canonical and non-canonical TGF-ß pathways.

Staurosporine has been known to induce chondrogenesis in monolayer cultures of mesenchymal cells by dissolving actin stress fibers. The aim of this study was to further elucidate how the alteration of actin filaments by staurosporine induces chondrogenesis. Specifically, we examined whether the transforming growth factor (TGF)-ß pathway is implicated. SB505124 strongly suppressed staurosporine-induced chondrogenesis without affecting the drug's action on the actin cytoskeleton. Staurosporine increased the phosphorylation of TGF-ß receptor I (TßRI) but had no significant effect on the expression levels of TGF-ß1, TGF-ß2, TGF-ß3, TßRI, TßRII, and TßRIII. Phosphorylation of Smad2 and Smad3 was not increased by staurosporine. However, SB505124 almost completely suppressed the phosphorylation of Smad2 and Smad3. In addition, inhibition of Smad3 blocked staurosporine-induced chondrogenesis. Inhibition of Akt, p38 mitogen-activated protein kinase (MAPK), and c-jun N-terminal kinase (JNK) suppressed chondrogenesis induced by staurosporine. Phosphorylation of Akt, p38 MAPK, and JNK was increased by staurosporine. SB505124 reduced the phosphorylation of Akt and p38 MAPK, while it had no effect on the phosphorylation of JNK. The phosphorylation level of extracellular signal-regulated kinase (ERK) was not significantly affected by staurosporine. In addition, inhibition of ERK with PD98059 alone did not induce chondrogenesis. Taken together, these results suggest that staurosporine induces chondrogenesis through TGF-ß pathways including canonical Smads and non-canonical Akt and p38 MAPK signaling.

Identification of a novel nonsynonymous mutation of EYA1 disrupting splice site in a Korean patient with BOR syndrome.

The EYA1 gene is known as the causative gene of BOR (Branchio-oto-renal) syndrome which is a genetic disorder associated with branchial cleft cysts of fistulae, hearing loss, ear malformation, and renal anomalies. Although approximately 40% of patients with BOR syndrome have mutations in the EYA1 gene and over 130 disease-causing mutations in EYA1 have been reported in various populations, only a few mutations have been reported in Korean families. In this study, genetic analysis of the EYA1 gene was performed in a Korean patient diagnosed with BOR syndrome and his parents. A de novo novel missense mutation, c.418G>A, located at the end of exon 6, changed glycine to serine at amino acid position 140 (p.G140S) and was suspected to affect normal splicing. Our in vitro splicing assay demonstrated that this mutation causes exon 6 skipping leading to frameshift and truncation of the protein to result in the loss of eyaHR. To the best of our knowledge, this is the first report revealing that a missense mutation in the exon disturbs normal splicing as a result of a substitution of the last nucleotide of an exon in EYA1.

Protein kinase Cα protects against multidrug resistance in human colon cancer cells.

Multidrug resistance is the phenomenon by which, after exposure to a single chemotherapeutic agent, cancer cells evade the agent's cytotoxic effects as well as become resistant to several classes of diverse drugs. ATP-binding cassette (ABC) transporters are a family of transporter proteins that contribute to drug resistance via a n ATP - dependent drug efflux pump. P-glycoprotein (P-gp) is a prominent ABC superfamily protein encoded by the mdr gene which has the ability to mediate the cellular extrusion of xenobiotics and anticancer drugs from tumor cells. Exclusively expressed P-gp cells from the human colon cancer HCT15/DOX line showed resistance to doxorubicin while parental HCT15 cells treated with doxorubicin displayed typical signs of apoptosis. In order to verify the hypothesis that expression of MDR is controlled in part, by protein kinase C (PKC), expression patterns of different PKC isoforms were examined in both cell lines. Of the PKC isoforms evaluated, the membrane translocation and expression levels of PKCα were strikingly increased in HCT15/DOX cells. PKCα reversed doxorubicin-induced apoptosis through the scavenging of ROS as well as inhibition of PARP cleavage. In addition, inhibition of PKCα with Go6976, a specific inhibitor of classical PKC, led to reduced MDR expression and increased doxorubicin-induced apoptosis. Knockdown of PKCα by siRNA diminished the protective effects of PKCα for doxorubicin-induced apoptosis. These results suggested that over-expression and activity of PKCα is closely associated with the regulation of the MDR phenotype in human colon cancer HCT15 cells and provided insight into a new strategy for inhibiting doxorubicin resistance in human cancers.

Inhibition of RhoA but not ROCK induces chondrogenesis of chick limb mesenchymal cells.

Cell shape change and cytoskeletal reorganization are known to be involved in the chondrogenesis. Negative role of RhoA, a cytoskeleton-regulating protein, and its downstream target, Rho-associated protein kinase (ROCK) in the chondrogenesis has been studied in many different culture systems including primary chondrocytes, chondrogenic cell lines, dedifferentiated chondrocytes, and micromass culture of mesenchymal cells. To further investigate the role of RhoA and ROCK in the chondrogenesis, we examined the RhoA-ROCK-myosin light chains (MLC) pathway in low density culture of chick limb bud mesenchymal cells. We observed for the first time that inhibition of RhoA by C3 cell-permeable transferase, CT04, induced chondrogenesis of undifferentiated mesenchymal single cells following dissolution of actin stress fibers. Inhibition of RhoA activity by CT04 was confirmed by pull down assay using the Rho-GTP binding domain of Rhotekin. CT04 also inhibited ROCK activity. In contrast, inhibition of ROCK by Y27632 neither altered the actin stress fibers nor induced chondrogenesis. In addition, inhibition of RhoA or ROCK did not affect the phosphorylation of MLC. Inhibition of myosin light chain kinase (MLCK) by ML-7 or inhibition of myosin ATPase with blebbistatin dissolved actin stress fibers and induced chondrogenesis. ML-7 reduced the MLC phosphorylation. Taken together, our current study suggests that RhoA uses other pathway than ROCK/MLC in the modulation of actin stress fibers and chondrogenesis. Our data also imply that, irrespective of mechanisms, dissolution of actin stress fibers is crucial for chondrogenesis.

TGF-beta3 inhibits chondrogenesis by suppressing precartilage condensation through stimulation of N-cadherin shedding and reduction of cRREB-1 expression.

Transforming growth factor-beta (TGF-beta) plays crucial roles in controlling cell differentiation and maintaining tissue integrity. Previously we reported that TGF-beta3 treatment decreased the mRNA expression of the gap junction protein, connexin 43 as well as cell number, which lead to the inhibition of chondrogenic condensation in cultured chick leg bud mesenchymal cells. The present study demonstrates that TGF-beta3 can induce cleavage in the ectodomain of neuronal cadherin (N-cadherin) at the initiation stage of chondrogenesis and reduce cell numbers, cellular adhesion and the expression level of connexin 43. Differential displayed PCR (DD-PCR) comparison of adherent- and non-adherent chick leg chondrogenic progenitor cells showed increased expression of the chick ras-responsive element binding transcription factor, cRREB-1, in adherent cells. In chick leg bud mesenchymal cells, cRREB-1 transcription was inhibited by TGF-beta3 at the early stage of chondrogenesis. Small interfering RNA (siRNA)-mediated knockdown of cRREB-1 reduced cell numbers, cellular adhesion, and the expression level of connexin 43 resulting in the inhibition of precartilage condensation. Taken together, these findings indicate that TGF-beta3 mediates the inhibitory signal necessary for precartilage condensation by stimulating N-cadherin shedding and reducing cRREB-1 expression levels.

Distribution of TGF-beta isoforms and signaling intermediates in corneal fibrotic wound repair.

In this study, temporal and spatial distribution of three TGF-beta isoforms and their downstream signaling pathways including pSmad2 and p38MAPK were examined during fibrotic wound repair. In normal chick corneas, TGF-beta1, -2, and -3 were weakly detected in Bowman's layer (BL). In healing corneas, TGF-beta1 was primarily deposited in the fibrin clot and the unwounded BL. TGF-beta2 was highly expressed in healing epithelial and endothelial cells, and numerous active fibroblasts/myofibroblasts. TGF-beta3 was mainly detected in the unwound region of basal epithelial cells. alpha-Smooth muscle actin (alpha-SMA) was initially appeared in the posterior region of repairing stroma at day 3, and was detected in the entire healing stroma by day 7. Notably, alpha-SMA was absent in the central region of healing stroma by day 14, and its staining pattern was similar to those of TGF-beta2 and p38MAPK. By contrast, pSmad2 was mainly detected in the fibroblasts. In normal cornea, laminin was mainly detected in both epithelial basement membrane (BM) and Descemet's membrane (DM). By contrast to reconstitution of the BM in the wound region, the DM was not repaired although endothelial layer was regenerated, indicating that high levels of TGF-beta2 were released into the posterior region of healing stroma on day 14. High levels of alpha-SMA staining, shown in cultured repair stromal cells from healing corneas on day 14 and in TGF-beta2 treated normal stromal cells, were significantly reduced by p38MAPK inhibition. Collectively, this study suggests that TGF-beta2-mediated myofibroblast transformation is mediated, at least partly, by the p38MAPK pathway in vivo.

Integrin signaling and cell spreading alterations by rottlerin treatment of chick limb bud mesenchymal cells.

Endochondral skeletal development begins with the formation of a cartilaginous template where mesenchymal cells aggregate and increase in density prior to their overt differentiation into chondrocytes. Prechondrogenic condensation, in which mesenchymal cells aggregate, requires cell migration and proliferation. However, the molecular mechanisms promoting this aggregation remain to be elucidated. Here, we report that rottlerin suppresses migration and cell surface expression of integrin beta1 in chondrogenic progenitors. Perturbation of integrin beta1 function using an anti-integrin beta1 blocking antibody suppressed the migration of wing bud mesenchymal cells. Furthermore, phosphorylation levels of Src and focal adhesion kinase (FAK) were decreased by rottlerin treatment. Cell treatment with PP2, an inhibitor of Src family kinase, or electroporation of FAK specific siRNA, suppressed cell migration in a wound-healing assay. Cells treated with rottlerin showed decreased phosphorylation of Akt, independent of PKCdelta inhibition. In addition, an Akt inhibitor suppressed the migration of chick limb bud mesenchymal cells. Taken together, our results point to the novel finding that rottlerin may act as a negative regulator for cell migration, an essential step for prechondrogenic condensation, by regulating integrin beta1 signaling at focal adhesion complexes via modulation of Akt activity.