Charleata A. Carter

Platelet-rich plasma gel promotes differentiation and regeneration during equine wound healing

Nonhealing wounds of the lower equine limb represent a challenging model. The platelet is a natural source of a myriad of growth factors and cytokines that promote wound healing. This study evaluates the potential of platelet derived factors to enhance wound healing in the lower equine limb. Platelets were isolated from horse blood and activated with thrombin, a process known to induce growth factor release. This produced a platelet gel composed of platelet-rich plasma (PRP). To test this all-natural wound healant, 2.5-cm(2) full thickness cutaneous wounds were created below the knee and hock of a thoroughbred horse. Wounds were treated with PRP gel or left untreated. Sequential wound biopsies collected at Days 7, 36, and 79 postwounding permitted comparison of the temporal expression of differentiation markers and wound repair. To test the hypothesis that wounds treated with PRP gel exhibit more rapid epithelial differentiation and enhanced organization of dermal collagen compared to controls, tissues were stained for cytokeratin 10, a suprabasal differentiation marker, and the reestablishment of collagen was evaluated by trichrome staining. PRP gel-treated wounds at Day 7 expressed intense cytokeratin 10 staining near the wound junction in suprabasal epidermal layers, while staining in control tissues was less intense and restricted to apical epidermal layers distal to the wound junction. By Day 79, the staining was equal in both groups. However, PRP gel-treated wounds at Day 79 contained abundant, dense collagen bundles oriented parallel to each other and to the overlying epithelium, whereas control tissues contained fewer collagen fibers that were oriented randomly. Thus, treatment of wounds with PRP gel induced accelerated epithelial differentiation and produced tissue with organized, interlocking collagen bundles. This study reveals that this novel all-natural wound healant induced wound repair in injuries previously deemed untreatable.

Direct Interaction of All-trans-retinoic Acid with Protein Kinase C (PKC) IMPLICATIONS FOR PKC SIGNALING AND CANCER THERAPY

Protein kinase C (PKC) regulates fundamental cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. All-trans-retinoic acid (atRA) modulates PKC activity, but the mechanism of this regulation is unknown. Amino acid alignments and crystal structure analysis of retinoic acid (RA)-binding proteins revealed a putative atRA-binding motif in PKC, suggesting existence of an atRA binding site on the PKC molecule. This was supported by photolabeling studies showing concentration- and UV-dependent photoincorporation of [3H]atRA into PKCα, which was effectively protected by 4-OH-atRA, 9-cis-RA, and atRA glucuronide, but not by retinol. Photoaffinity labeling demonstrated strong competition between atRA and phosphatidylserine (PS) for binding to PKCα, a slight competition with phorbol-12-myristate-13-acetate, and none with diacylglycerol, fatty acids, or Ca2+. At pharmacological concentrations (10 μm), atRA decreased PKCα activity through the competition with PS but not phorbol-12-myristate-13-acetate, diacylglycerol, or Ca2+. These results let us hypothesize that in vivo, pharmacological concentrations of atRA may hamper binding of PS to PKCα and prevent PKCα activation. Thus, this study provides the first evidence for direct binding of atRA to PKC isozymes and suggests the existence of a general mechanism for regulation of PKC activity during exposure to retinoids, as in retinoid-based cancer therapy.

Therapeutic potential of natural compounds that regulate the activity of protein kinase C.

Protein kinase C (PKC) is a family of serine/threonine kinases that regulates a variety of cell functions including proliferation, gene expression, cell cycle, differentiation, cytoskeletal organization, cell migration, and apoptosis. The PKC signal transduction cascade coordinates complex physiological events including normal tissue function and repair. Disruption of the cellular environment through genetic mutation, disease, injury, or exposure to pro-oxidants, alcohol, or other insults can induce pathological PKC activation. Aberrant PKC activation can lead to diseases of cellular dysregulation such as cancer and diabetes. Can aberrant activation of PKC be reversed? Even 25 years after the identification of PKC, therapeutic regulation of PKC activity remains an emerging field. Because the function of each isoform remains to be elucidated, isoform specific control of gene expression is a current challenge. Natural compounds are important regulators of PKC activity, with both preventive and therapeutic efficacy. Antioxidants including vitamin A (retinoids), vitamin C (ascorbic acid) and vitamin E (tocopherols) show promise for reversal of PKC activation. beta-carotene and retinoids function as anticarcinogenic agents and antagonize the biological effects of pro-oxidants on PKC. Vitamin E reverses the deleterious effects of hyperglycemia and diabetes by down-regulating PKC activity. Antioxidants in red wine provide cardioprotective effects. However, alcohol consumption also induces oxidative stress and disrupts PKC and retinoid function in the fetus and the adult. This review examines modulation of PKC activity by natural compounds and pharmacologic analogues which can be used effectively to prevent or treat common diseases associated with aberrant activation of PKC.

Cytoskeletal Reorganization Induced by Retinoic Acid Treatment of Human Endometrial Adenocarcinoma (RL95-2) Cells Is Correlated with Alterations in Protein Kinase C-α

We have shown previously that treatment of human endometrial adenocarcinoma (RL95-2) cells with either 13-cis or all-trans retinoic acid results in reorganization of actin filaments, indicating reversion to a stationary phenotype. In the present study, we investigated the role of protein kinase C (PKC) in this process. Treatment of cells with PKC inhibitors (staurosporine, bisindolylmaleimide, or Gö6976) resulted in morphological alterations and reorganization of actin filaments similar to retinoic-acid-treated cells. For example, RL95-2 cells treated with staurosporine flattened, exhibited cell surface extensions and some actin filaments. Bisindolylmaleimide-treated cells flattened, and actin filaments reorganized similar to retinoic-acid-treated cells. RL95-2 cells treated with Gö6976, which inhibits only PKCα, β, and γ, exhibited many cell surface extensions and some actin filament reorganization. We then investigated whether retinoic acid affected the subcellular localization of PKC-α. In control cells, PKC-α was mainly evident as diffuse cytoplasmic immunostaining, with a small percentage of total PKC-α also evident in the plasma membrane. Retinoic acid treatment dramatically altered PKC-α localization, since a more distinct cytoplasmic and perinuclear staining pattern was apparent. Western blot analysis confirmed these results, since the amount of cytosolic PKC-α increased following retinoic acid treatment. Thus, retinoic-acid-induced endometrial differentiation may be associated with alterations in PKC-α localization and signaling.

Retinoic acid signaling through PI 3-kinase induces differentiation of human endometrial adenocarcinoma cells.

The specific signals required for actin polymerization in response to extracellular factors remain unknown. However, in many cell types, there is a correlation between actin polymerization, activation of phosphatidylinositol 3-kinase (PI 3-kinase), and the production of the second messenger phosphatidylinositol-3,4,5-triphosphate. Increased levels of PI 3-kinase have been detected during cell growth and transformation. However, PI 3-kinase is also activated during differentiation, suggesting that PI 3-kinase and its lipid products also play a role in the regulation of cellular differentiation. The newly characterized CAC-1 cell line established from a poorly differentiated human endometrial adenocarcinoma (Exp. Mol. Pathol. 69 (2000), 175) was used as a model to investigate the role of PI 3-kinase in differentiation induction. CAC-1 cells differentiated upon treatment with pharmacological doses of retinoids (1 micro M of 13-cis or all-trans), evidenced by actin filament reorganization, and cell enlargement. PI 3-kinase staining is primarily localized to perinuclear regions in untreated cells. However, retinoic acid treatment induced PI 3-kinase to relocalize throughout the cytoplasm. Subcellular fractionation and Western blotting confirmed that PI 3-kinase decreased in the particulate fraction, concurrent with retinoid-induced differentiation. Interestingly, pretreatment with the PI 3-kinase inhibitor wortmannin (100 nM) prior to retinoic acid treatment prevented retinoic acid-induced actin reorganization and cell enlargement. To distinuish whether retinoid regulation of PI 3-kinase is mediated through traditional nuclear retinoic acid receptors, the levels of retinoic acid receptor-beta (RAR-beta) protein were evaluated. Retinoid treatment did not alter RAR-beta protein levels compared to controls. These data suggest that PI 3-kinase activity and cytoplasmic relocalization are required for retinoid-induced differentiation of poorly differentiated human endometrial adenocarcinoma cells.

Decrease in protein tyrosine phosphorylation is associated with F‐actin reorganization by retinoic acid in human endometrial adenocarcinoma (RL95‐2) cells

Transformed cells often express elevated levels of tyrosine‐phosphorylated proteins. Inhibition of protein tyrosine kinases causes reversion of malignant cells to the normal phenotype. In the present study, we evaluated the possibility that the reversion of human endometrial adenocarcinoma RL95‐2 cells to a stationary phenotype induced by retinoic acid was associated with inhibition of tyrosine phosphorylation of cellular proteins. We found that retinoic acid decreased the levels of tyrosine‐phosphorylated proteins, as assessed by immunostaining and immunoprecipitations using specific anti‐phosphotyrosine antibodies. In addition, the inhibitors of tyrosine kinases herbimycin A and tyrphostin mimicked retinoic acid, inducing F‐actin reorganization and increasing the size of RL95‐2 cells, as determined by measurement of cell perimeters. Because focal adhesions that connect actin filaments with the plasma membrane are major sites of tyrosine phosphorylation, we further investigated whether selected focal adhesion proteins were affected by retinoic acid. We found that retinoic acid altered the localization of focal adhesion kinase. All‐trans retinoic acid was effective in reducing the levels of focal adhesion kinase and paxillin protein. Thirteen‐cis retinoic acid increased the levels of vinculin protein in the cytosolic fraction of cells. These changes are consistent with actin reorganization and reversion toward a stationary phenotype induced by retinoic acid in endometrial adenocarcinoma RL95‐2 cells. Our results indicate that the differentiating effects of retinoids on endometrial cells are associated with decreases in tyrosine phosphorylation and changes in the levels and distribution of focal adhesion proteins. These findings suggest that signaling pathways that involve tyrosine kinases are potential targets for drug design against endometrial cancer. J. Cell. Physiol. 178:320–332, 1999.

Multiple cellular proteins are recognized by the adeno-associated virus Rep78 major regulatory protein and the amino-half of Rep78 is required for many of these interactions

Adeno‐associated virus (AAV) encoded Rep78 is a multi‐functional protein which is required for AAV DNA replication, is able to regulate both AAV and heterologous gene expression at the transcriptional level, and appears necessary for site specific integration of AAV DNA into human chromosome 19. By comparison with the analogous replication protein of the polyomaviruses, large T antigen, it seemed likely that Rep78 would interact with cellular proteins to carry out at least some its functions. This study demonstrates that Rep78 is able to interact with multiple cellular proteins from cellular extracts as measured by West(far)‐western, coimmunoprecipitation, and Rep78‐affinity chromatography analysis. Eight cellular proteins of approximately 180, 140, 120, 95, 75, 55, 45, and 35 kDa (+/‐10%), were observed to bind Rep78 in all three assay systems. Two others, of 30 and 24 kDa, were observed in two of three assay systems. Furthermore, using truncated Rep78 proteins, it is demonstrated that the amino‐terminus is required for most Rep78‐cellular protein interactions. However, the extreme carboxy‐terminus of Rep78 was found to be all that is required for binding to the 55 kDa cellular protein.

Tamoxifen alters the localization of F-actin and alpha 5/beta 1-integrin fibronectin receptors in human endometrial stromal cells and carcinoma cells.

We have investigated F-actin and the integrin fibronectin receptor as possible targets of tamoxifen (TAM) signaling in a cell-based model of the endometrium. Normal human endometrial stromal cells and RL95-2 human endometrial adenocarcinoma cells were treated for 1 h with TAM, a known antagonist of protein kinase C (PKC), or with staurosporine or HA1004, two broad-spectrum protein kinase antagonists capable of inhibiting PKC and PKA, respectively. We utilized fluorescein-phalloidin and confocal microscopy to visualize the cellular distribution of F-actin. Normal stromal cells and RL95-2 cells differed in the arrangement of F-actin in control cells and in their response to TAM. In control stromal cells, actin stress fibers were well organized throughout the cell, but in RL95-2 cells, they were disorganized and present mainly at the cell periphery. F-actin in RL95-2 cells treated with TAM (0.1 and 1.0 microM) or with staurosporine (0.7 and 7.0 nM) exhibited a reorganization into stress fibers consistent with a more stationary phenotype. In contrast, TAM- or staurosporine-treated normal stromal cells exhibited an increase in the amount of organized F-actin. Interestingly, in normal stromal cells treated with staurosporine but not TAM or HA1004, these F-actin fibers appeared to terminate in dense plaques proximal to the plasma membrane. The alpha 5/beta 1 integrin fibronectin receptor mediates between the extracellular matrix and the actin cytoskeleton. TAM induced clustering of the fibronectin receptor at the plasma membrane in normal stromal cells, but not in carcinoma cells. This study supports the importance of plasma membrane-cytoskeletal protein interactions in the response of normal and carcinoma cells to TAM.