The role of integrin-linked kinase in liver wound healing

Liver wound healing is an integrated process in which hepatic stellate cells play a major role. We hypothesized that the cellextracellular signaling protein integrin-linked kinase (ILK) is important in transducing signals from the extracellular matrix to stellate cells and thus plays a critical role in stellate cell activation and fibrogenesis during liver injury. Liver injury and subsequent stellate cell activation led to a 3-fold increase in ILK expression and increased kinase activity. Overexpression of ILK in isolated stellate cells led to enhanced motility and adhesion as well as increases in smooth muscle alpha-actin and type I collagen mRNA expression. The effects of ILK on stellate cell phenotypes were phosphatidylinositol 3-kinase-dependent. Forced expression of ILK in vivo led to increases in type I collagen, smooth muscle alpha-actin, transforming growth factor-beta, and extra domain A (EDA) fibronectin mRNAs (by 3.2-, 3.5-, 2.5-, and 2.2-fold, respectively; n = 8, p < 0.05 for each versus the control), whereas inhibition of ILK in vivo led to significant reductions in these mRNAs. Morphometric analysis revealed that ILK overexpression led to a 31.4% increase in liver collagen content (n = 8, p < 0.05 versus the control); in contrast ILK knockdown in vivo led to a significant reduction in fibrogenesis. We conclude that ILK plays an important pathophysiological role in vivo in liver wound healing.     

Wound-induced contractile ring: a model for cytokinesis.

The actomyosin-based contractile ring is required for several biological processes, such as wound healing and cytokinesis of animal cells. Despite progress in defining the roles of this structure in both wound closure and cell division, we still do not fully understand how an actomyosin ring is spatially and temporally assembled, nor do we understand the molecular mechanism of its contraction. Recent results have demonstrated that microtubule-dependent local assembly of F-actin and myosin-II is present in wound closure and is similar to that in cytokinesis in animal cells. Furthermore, signalling factors such as small Rho GTPases have been shown to be involved in the regulation of actin dynamics during both processes. In this review we address recent findings in an attempt to better understand the dynamics of actomyosin contractile rings during wound healing as compared with the final step of animal cell division.     

Functional analysis of a human homologue of the Drosophila actin binding protein anillin suggests a role in cytokinesis

We have characterized a human homologue of anillin, a Drosophila actin binding protein. Like Drosophila anillin, the human protein localizes to the nucleus during interphase, the cortex following nuclear envelope breakdown, and the cleavage furrow during cytokinesis. Anillin also localizes to ectopic cleavage furrows generated between two spindles in fused PtK(1) cells. Microinjection of antianillin antibodies slows cleavage, leading to furrow regression and the generation of multinucleate cells. GFP fusions that contain the COOH-terminal 197 amino acids of anillin, which includes a pleckstrin homology (PH) domain, form ectopic cortical foci during interphase. The septin Hcdc10 localizes to these ectopic foci, whereas myosin II and actin do not, suggesting that anillin interacts with the septins at the cortex. Robust cleavage furrow localization requires both this COOH-terminal domain and additional NH(2)-terminal sequences corresponding to an actin binding domain defined by in vitro cosedimentation assays. Endogenous anillin and Hcdc10 colocalize to punctate foci associated with actin cables throughout mitosis and the accumulation of both proteins at the cell equator requires filamentous actin. These results indicate that anillin is a conserved cleavage furrow component important for cytokinesis. Interactions with at least two other furrow proteins, actin and the septins, likely contribute to anillin function.     

Peroxynitrite activates mitogen-activated protein kinase (MAPK) via a MEK-independent pathway: a role for protein kinase C.

In this study we show that phosphorylation of extracellular signal-regulated kinase (ERK1/2; also known as p44/42MAPK) following peroxynitrite (ONOO(-)) exposure occurs via a MAPK kinase (MEK)-independent but PKC-dependent pathway in rat-1 fibroblasts. ONOO(-)-mediated ERK1/2 phosphorylation was not blocked by MEK inhibitors PD98059 and U0126. Furthermore, no increase in MEK phosphorylation was detected upon ONOO(-) treatment. Staurosporine was used to investigate whether protein kinase C (PKC) is involved. This was confirmed by down-regulation of PKC by phorbol-12,13-dibutyrate, which resulted in significant reduction of ERK1/2 phosphorylation by ONOO(-), implying that activation of ERK by ONOO(-) depends on activation of PKC. Indeed, PKCalpha and epsilon were activated upon ONOO(-) exposure. When cells were treated with ONOO(-) in a calcium-free buffer, no activation of PKCalpha was detected. Concomitantly, a reduction of ERK1/2 phosphorylation was observed suggesting that calcium was required for translocation of PKCalpha and ERK phosphorylation by ONOO(-). Indeed, ONOO(-) exposure resulted in increased cytosolic calcium, which depended on the presence of extracellular calcium. Finally, data using G?6976, an inhibitor of calcium-dependent PKC activation, implied that ONOO(-)-mediated ERK1/2 phosphorylation depends on activation of a calcium-dependent PKC.     

A role for protein kinase C in associative learning

Recent work suggests that protein kinase C (PKC), an enzyme that has a critical role in the regulation of cell growth and differentiation, also participates in the sequence of molecular events that underlie learning and memory. By means of electrophysiological, biochemical, and neuro-imaging methods it has been demonstrated that, in the brain, the distribution of PKC changes as a result of memory storage. The changes in distribution occur within the same ensembles of nerve cells that are necessary for the acquisition and performance of various learning tasks in several species. Here we review the data pertaining to a model that has been proposed to account for the participation of PKC as a molecular signal for cotemporal synaptic input during associative learning.     

Activation of protein kinase C isozymes by contractile stimuli in arterial smooth muscle.

Protein kinase C (PKC) has been proposed to be involved in the regulation of vascular smooth muscle (VSM) contractile activity. However, little is known in detail about the activation of this kinase or specific isozymes of this kinase by contractile stimuli in VSM. As an index of PKC activation, Ca(2+)- and phospholipid-dependent histone IIIS kinase activity was measured in the particulate fraction from individual strips of isometrically contracting carotid arterial smooth muscle. Phorbol 12,13-dibutyrate (PDB) increased PKC activity in the particulate fraction (155% over resting value by 15 min) with a time course which paralleled or preceded force development. Stimulation with the agonist histamine (10(-5) M) resulted in rapid increases in both force and particulate fraction PKC activity which was maximal by 2 min (increase of 139%) and partially sustained over 45 min (increase of 41%). KCl (109 mM), which evokes a sustained contractile response, caused a slow increase (124% by 45 min) in particulate fraction PKC activity. No significant increases in activator-independent histone kinase activity were observed in response to any stimulus tested. PKC alpha and PKC beta were identified as the principal Ca2+/phospholipid-dependent PKC isozymes expressed in this tissue. In unstimulated arterial tissue, the ratio of immunodetectable isozyme content (alpha:beta) was estimated to be 1:1 in the particulate and 1.5:1 in the cytosolic fractions. Upon stimulation with each of the three contractile stimuli, particulate fraction PKC content assessed by immunoblotting increased with a time course and to an extent comparable to the observed changes in PKC activity. There was no evidence of differential regulation of the PKC alpha or -beta isozymes by PDB compared to the other contractile stimuli. These results indicate that diverse contractile stimuli are capable of tonically activating PKC in preparations of functional smooth muscle, and are consistent with a functional role for PKC alpha and/or -beta in the regulation of normal smooth muscle contractile activity.     

An essential role for a membrane lipid in cytokinesis. Regulation of contractile ring disassembly by redistribution of phosphatidylethanolamine

Phosphatidylethanolamine (PE) is a major membrane phospholipid that is mainly localized in the inner leaflet of the plasma membrane. We previously demonstrated that PE was exposed on the cell surface of the cleavage furrow during cytokinesis. Immobilization of cell surface PE by a PE-binding peptide inhibited disassembly of the contractile ring components, including myosin II and radixin, resulting in formation of a long cytoplasmic bridge between the daughter cells. This blockade of contractile ring disassembly was reversed by removal of the surface-bound peptide, suggesting that the PE exposure plays a crucial role in cytokinesis. To further examine the role of PE in cytokinesis, we established a mutant cell line with a specific decrease in the cellular PE level. On the culture condition in which the cell surface PE level was significantly reduced, the mutant ceased cell growth in cytokinesis, and the contractile ring remained in the cleavage furrow. Addition of PE or ethanolamine, a precursor of PE synthesis, restored the cell surface PE on the cleavage furrow and normal cytokinesis. These findings provide the first evidence that PE is required for completion of cytokinesis in mammalian cells, and suggest that redistribution of PE on the cleavage furrow may contribute to regulation of contractile ring disassembly.     

Platelet-derived growth factor-induced c-myc RNA expression. Analysis of an inducible pathway independent of protein kinase C

Platelet-derived growth factor (PDGF) is generally considered to stimulate phosphoinositide turnover resulting in activation of protein kinase C and increased cytoplasmic [Ca2+]. We have examined the role of these secondary effects in regulation of c-myc mRNA accumulation in the MG-63 human osteogenic sarcoma line. Treatment of quiescent cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA) to down-regulate protein kinase C inhibited TPA-stimulated c-myc expression but did not affect the PDGF-modulated process. When cytoplasmic [Ca2+] was increased by addition of a Ca2+ ionophore (A23187 or ionomycin), no stimulation of c-myc RNA was seen; furthermore, these agents did not enhance the PDGF-modulated c-myc expression. Addition of EGTA to cultures treated with both PDGF and a Ca2+ ionophore did not inhibit c-myc induction but rather caused a superinduction of c-myc RNA accumulation. Superinduction occurred only if the [EGTA] was greater than [Ca2+] in the medium. This superinduction was distinct from the increased induction caused by inhibition of protein synthesis. Because PDGF-induced c-myc expression is independent of protein kinase C and increased cytoplasmic [Ca2+], the evidence suggests that PDGF modulates c-myc RNA accumulation in MG-63 cells via a novel pathway, seemingly uncoupled from the classic action of increased phosphoinositide metabolism.     

B cell activation. IV. Induction of cell membrane depolarization and hyper-I-A expression by phorbol diesters suggests a role for protein kinase C in murine B lymphocyte activation

Analysis of the effects of phorbol diesters on mouse B lymphocyte kinase C activity, membrane potential, mI-A expression, and cell cycle state are reported. Results indicate that the phorbol diesters PMA and 4 beta-PDD, which are potent tumor promoters, activate partially purified B cell protein kinase C and stimulate B cell membrane depolarization and increased mI-A expression. The analog 4 alpha-PDD has none of these effects. Similarly, none of the phorbol diesters tested promoted G0 to G1 transition of B lymphocytes. Results are consistent with the possibility that the transmembrane signal transduction mediated by cell membrane immunoglobulin, which results in membrane depolarization and increased I-A antigen expression, operates via activation of protein kinase C.     

Structure of Cdc4p, a contractile ring protein essential for cytokinesis in Schizosaccharomyces pombe

The Schizosaccharomyces pombe Cdc4 protein is required for the formation and function of the contractile ring, presumably acting as a myosin light chain. By using NMR spectroscopy, we demonstrate that purified Cdc4p is a monomeric protein with two structurally independent domains, each exhibiting a fold reminiscent of the EF-hand class of calcium-binding proteins. Although Cdc4p has one potentially functional calcium-binding site, it does not bind calcium in vitro. Three variants of Cdc4p containing single point mutations responsible for temperature-sensitive arrest of the cell cycle at cytokinesis (Gly-19 to Glu, Gly-82 to Asp, and Gly-107 to Ser) were also characterized by NMR and circular dichroism spectroscopy. In each case, the amino acid substitution only leads to small perturbations in the conformation of the protein. Furthermore, thermal unfolding studies indicate that, like wild-type Cdc4p, the three mutant forms are all extremely stable, remaining completely folded at temperatures significantly above those causing failure of cytokinesis in intact cells. Therefore, the altered phenotype must arise directly from a disruption of the function of Cdc4p rather than indirectly through a disruption of its overall structure. Several mutant alleles of Cdc4p also show interallelic complementation in diploid cells. This phenomenon can be explained if Cdcp4 has more than one essential function or, alternatively, if two mutant proteins assemble to form a functional complex. Based on the structure of Cdc4p, possible models for interallelic complementation including interactions with partner proteins and the formation of a myosin complex with Cdc4p fulfilling the role of both an essential and regulatory light chain are proposed.     

The structure of Antirrhinum centroradialis protein (CEN) suggests a role as a kinase regulator

Expression of the plant protein centroradialis (CEN) leads to a morphological switch between shoot growth and the development of flower structures (inflorescence). We have determined the crystal structure of Antirrhinum CEN to 1.9 A resolution. This structure confirms the CEN proteins as a subset of the family of phosphatidylethanolamine-binding proteins (PEBP), as predicted from sequence homology. Mammalian forms of PEBP have been found to act as inhibitors of MAP kinase signalling, a central signalling cascade regulating cell differentiation. CEN and PEBP proteins share a similar topology dominated by a large central beta-sheet. The strong conservation of a binding pocket at one end of this sheet which is capable of binding phosphoryl ligands, suggests the biological effects of CEN, like PEBP, arise from the ability of this region to form complexes with phosphorylated ligands, hence interfering with kinases and their effectors.     

A role for calcium and protein kinase C in agonist-stimulated adhesion of human neutrophils.

Stimulated adherence of human neutrophils to plastic and changes in cytosolic free Ca2+ concn. [( Ca2+]i) were measured in the same cell preparations. [Ca2+]i-activation curves were constructed to compare the relation between [Ca2+]i and adhesion in response to ionomycin and formylmethionyl-leucyl-phenylalanine (FMLP). This showed that FMLP-stimulated adhesion required less increase in [Ca2+]i than did ionomycin's effect, a result suggesting that an additional stimulatory component might be involved in the response to FMLP. Protein kinase C activation was a possibility, and activation of protein kinase C with a phorbol ester (PMA) was found to stimulate adhesion with no change in [Ca2+]i. A low concentration of PMA was found to synergize with ionomycin to stimulate a greater adhesion response than with each alone, and the [Ca2+]i-activation curve for ionomycin in the presence of PMA was shifted towards that for FMLP. Thus, synergy between [Ca2+]i and protein kinase C (each of which is sufficient alone) probably explains the stimulatory effects of FMLP on adhesion of neutrophils.     

Evidence for a role of phosphatidylcholine-hydrolysing phospholipase C in the regulation of protein kinase C by ras and src oncogenes.

The products of ras and src oncogenes are thought to be important components in pathways regulating cell proliferation and differentiation. In fibroblasts transformed by these oncogenes, increased diacylglycerol levels have been found which most probably arise from activation of the turnover of phosphatidylcholine. Diacylglycerol is a key activator of protein kinase C whose role in cell growth and transformation has been proposed. We demonstrate here by using immunochemical techniques that transformation by ras or src oncogenes is associated with permanent translocation of protein kinase C to the cytoplasmic membrane. However, no down-regulation of the enzyme is observed despite its permanent activation in these transformants. Importantly, the lack of down-regulation observed in ras and src transformed cell lines is mimicked by chronic treatment of NIH 3T3 fibroblasts with exogenous Bacillus cereus phosphatidylcholine-hydrolysing phospholipase C, but not with phorbol myristate acetate or exogenous Bacillus thuringiensis phosphatidylinositol-hydrolysing phospholipase C. These results strongly suggest that diacylglycerol derived from phosphatidylcholine but not from phosphoinositide turnover is responsible for the atypical regulation of protein kinase C in cell lines transformed by ras and src oncogenes.     

Evidence for a role of protein kinase C zeta subspecies in maturation of Xenopus laevis oocytes.

A number of studies have demonstrated the activation of phospholipase C-mediated hydrolysis of phosphatidylcholine (PC-PLC) both by growth factors and by the product of the ras oncogene, p21ras. Evidence has been presented indicating that the stimulation of this phospholipid degradative pathway is sufficient to activate mitogenesis in fibroblasts as well as that it is sufficient and necessary for induction of maturation in Xenopus laevis oocytes. However, the mechanism whereby PC-PLC transduces mitogenic signals triggered by growth factors or oncogenes remains to be elucidated. In this study, data are presented that show the involvement of protein kinase C zeta subspecies in the channelling of the mitogenic signal activated by insulin-p21ras-PC-PLC in Xenopus oocytes as well as the lack of a critical role of protein kinase C isotypes alpha, beta, gamma, delta, and epsilon in these pathways.     

MicroRNA 在皮肤创伤愈合中的作用

皮肤创伤愈合过程是一个复杂而连续的过程,这一过程需要多种细胞、多种因子的参与,涉及细胞增殖、细胞分化、细胞运动、细胞黏附等多个细胞生物学过程。 MicroRNA（ miRNA）是一类高度保守的非编码RNA,它通过靶向结合信使RNA（ mRNA）并使其降解或抑制其翻译,实现转录后基因表达调控。 miRNA作为基因表达的重要调控分子,几乎参与了机体所有的生理和病理过程。除了在皮肤发育中发挥重要的作用,还参与多种皮肤病、皮肤癌和皮肤创伤愈合过程的调节。主要总结了miRNA调控皮肤创伤愈合的研究进展。     

Microvascular remodeling and wound healing: A role for pericytes

Physiologic wound healing is highly dependent on the coordinated functions of vascular and non-vascular cells. Resolution of tissue injury involves coagulation, inflammation, formation of granulation tissue, remodeling and scarring. Angiogenesis, the growth of microvessels the size of capillaries, is crucial for these processes, delivering blood-borne cells, nutrients and oxygen to actively remodeling areas. Central to angiogenic induction and regulation is microvascular remodeling, which is dependent upon capillary endothelial cell and pericyte interactions. Despite our growing knowledge of pericyte-endothelial cell crosstalk, it is unclear how the interplay among pericytes, inflammatory cells, glia and connective tissue elements shape microvascular injury response. Here, we consider the relationships that pericytes form with the cellular effectors of healing in normal and diabetic environments, including repair following injury and vascular complications of diabetes, such as diabetic macular edema and proliferative diabetic retinopathy. In addition, pericytes and stem cells possessing "pericyte-like" characteristics are gaining considerable attention in experimental and clinical efforts aimed at promoting healing or eradicating ocular vascular proliferative disorders. As the origin, identification and characterization of microvascular pericyte progenitor populations remains somewhat ambiguous, the molecular markers, structural and functional characteristics of pericytes will be briefly reviewed.     

The role of protein kinase C in T lymphocyte proliferation. Existence of protein kinase C-dependent and -independent pathways

The mouse cytotoxic T cell clone (CTLL-2) was able to grow in the presence of culture medium supplemented only with transferrin, 2-mercaptoethanol, and recombinant interleukin 2 (IL-2). This lymphokine stimulated the synthesis of DNA in these cells. Similarly, phorbol esters, which activate protein kinase C, induced DNA synthesis in this clone. Furthermore, this later proliferation was not blocked by anti-IL-2 receptor antibodies, which inhibited IL-2-induced proliferation, suggesting that it was not indirectly due to the secretion of IL-2 by the cells. CTLL-2 cells pretreated with high doses of phorbol esters for 48 h down regulated protein kinase C and were depleted of this enzyme. This was shown by: 1) purification and in vitro assay of protein kinase C; 2) the lack of effect of phorbol esters in the stimulation of the Na+/H+ anti-porter which has been directly linked to the activation of protein kinase C. As expected, those protein kinase C-depleted cells no longer synthesized DNA and proliferated in response to phorbol esters. However, they proliferated identically to control cells in response to IL-2. Therefore, our results suggest two different pathways for T cell proliferation, one which involves protein kinase C and the other which does not.