Cell wounding activates phospholipase D in primary mouse keratinocytes.

Plasma membrane disruptions occur in mechanically active tissues such as the epidermis and can lead to cell death if the damage remains unrepaired. Repair occurs through fusion of vesicle patches to the damaged membrane region. The enzyme phospholipase D (PLD) is involved in membrane traffickiing; therefore, the role of PLD in membrane repair was investigated. Generation of membrane disruptions by lifting epidermal keratinocytes from the substratum induced PLD activation, whereas removal of cells from the substratum via trypsinization had no effect. Pretreatment with 1,25-dihydroxyvitamin D3, previously shown to increase PLD1 expression and activity, had no effect on, and a PLD2-selective (but not a PLD1-selective) inhibitor decreased, cell lifting-induced PLD activation, suggesting PLD2 as the isoform activated. PLD2 interacts functionally with the glycerol channel aquaporin-3 (AQP3) to produce phosphatidylglycerol (PG); however, wounding resulted in decreased PG production, suggesting a potential PG deficiency in wounded cells. Cell lifting-induced PLD activation was transient, consistent with a possible role in membrane repair, and PLD inhibitors inhibited membrane resealing upon laser injury. In an in vivo full-thickness mouse skin wound model, PG accelerated wound healing. These results suggest that PLD and the PLD2/AQP3 signaling module may be involved in membrane repair and wound healing.

Immobilization of a phosphonated analog of matrix phosphoproteins within cross-linked collagen as a templating mechanism for biomimetic mineralization.

Immobilization of phosphoproteins on a collagen matrix is important for the induction of intrafibrillar apatite mineralization. Unlike phosphate esters, polyphosphonic acid has no reactive sites for covalent binding to collagen amine groups. Binding of poly(vinyl phosphonic acid) (PVPA), a biomimetic templating analog of matrix phosphoproteins, to collagen was found to be electrostatic in nature. Thus, an alternative retention mechanism was designed for immobilization of PVPA on collagen by cross-linking the latter with carbodiimide (EDC). This mechanism is based on the principle of size exclusion entrapment of PVPA molecules within the internal water compartments of collagen. By cross-linking collagen with EDC, a zero length cross-linking agent, the sieving property of collagen is increased, enabling the PVPA to be immobilized within the collagen. The absence of covalent cross-linking between PVPA and collagen was confirmed by Fourier transform infrared spectroscopy. Based on these results, a concentration range for immobilized PVPA to template intrafibrillar apatite deposition was established and validated using a single layer reconstituted type I collagen mineralization model. In the presence of a polyacrylic acid-containing mineralization medium optimal intrafibrillar mineralization of the EDC-cross-linked collagen was achieved using 500 and 1000 µg ml⁻¹ PVPA. The mineralized fibrils exhibited a hierarchical order of intrafibrillar mineral infiltration, as manifested by the appearance of electron-dense periodicity within unstained fibrils. Understanding the basic processes in intrafibrillar mineralization of reconstituted collagen creates opportunities for the design of tissue engineering materials for hard tissue repair and regeneration.

The potential use of protein kinase D inhibitors for prevention/treatment of epidermal tumors.

BACKGROUND: The serine/threonine kinase protein kinase D (PKD) has been proposed to be a pro-proliferative, anti-differentiative signal in epidermal keratinocytes. Indeed, the phorbol ester tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA) induces biphasic PKD activation, which mirrors the biphasic response of initial differentiation followed by proliferation and tumor promotion seen in TPA-treated keratinocytes in vitro and epidermis in vivo. OBJECTIVE: Our objective was to test the idea that PKD's pro-proliferative and/or anti-differentiative effects in keratinocytes contribute to TPA-induced tumorigenesis. METHODS: Using western analysis and assays of keratinocyte proliferation and differentiation, we investigated the effect of inhibitors of PKD on keratinocyte function. RESULTS: We found that overexpression of a constitutively active PKD mutant increased, and of a dominant-negative PKD mutant decreased, keratinocyte proliferation. A recently described selective PKD inhibitor showed low potency to inhibit keratinocyte proliferation or PKD activation. Therefore, we tested the ability of known only relatively selective PKD inhibitors on keratinocyte function and protein kinase activation. H89 {N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinoline-sulfonamide}, a reported inhibitor of PKD and cAMP-dependent protein kinase, enhanced the effect of a differentiating agent on a marker of keratinocyte differentiation. Another reported non-selective PKD inhibitor, resveratrol stimulated differentiation and inhibited proliferation. The protein kinase C/PKD inhibitor Gö6976 blocked the increase in proliferation (as measured by DNA specific activity) induced by chronic TPA without affecting the initial TPA-elicited differentiation. CONCLUSION: Our results support the idea that relatively selective PKD inhibitors, such as Gö6976, H89 and resveratrol, might be useful for preventing/treating epidermal tumorigenesis without affecting keratinocyte differentiation.

Effect of vapor lock on root canal debridement by using a side-vented needle for positive-pressure irrigant delivery.

INTRODUCTION: This study examined the effect of vapor lock on canal debridement efficacy by testing the null hypothesis that there is no difference between a "closed" and an "open" system design in smear layer and debris removal by using a side-vented needle for irrigant delivery. METHODS: Roots in the closed system were sealed with hot glue and embedded in polyvinylsiloxane to restrict fluid flow through the apical foramen during cleaning and shaping. For the open system, the apical foramen was enlarged and connected to the external environment via a channel within the polyvinylsiloxane to permit unrestricted fluid flow. Smear and debris scores were evaluated by using scanning electron microscopy and analyzed by using Cochran-Mantel-Haenszel statistic. RESULTS: No difference in smear scores was detected between the 2 systems at all canal levels. Significant differences in debris scores between the 2 systems were found at each canal level: coronal (P < .001), middle (P < .001), and apical (P < .001). CONCLUSIONS: The null hypothesis was rejected; presence of an apical vapor lock effect adversely affects debridement efficacy. Thus, studies with unspecified or questionable mechanisms to restrict fluid flow through the apical foramen have to be interpreted with caution.

Angiotensin II-activated protein kinase D mediates acute aldosterone secretion.

Dysregulation of the renin-angiotensin II (AngII)-aldosterone system can contribute to cardiovascular disease, such that an understanding of this system is critical. Diacylglycerol-sensitive serine/threonine protein kinase D (PKD) is activated by AngII in several systems, including the human adrenocortical carcinoma cell line NCI H295R, where this enzyme enhances chronic (24h) AngII-evoked aldosterone secretion. However, the role of PKD in acute AngII-elicited aldosterone secretion has not been previously examined. In primary cultures of bovine adrenal glomerulosa cells, which secrete detectable quantities of aldosterone in response to secretagogues within minutes, PKD was activated in response to AngII, but not an elevated potassium concentration or adrenocorticotrophic hormone. This activation was time- and dose-dependent and occurred through the AT1, but not the AT2, receptor. Adenovirus-mediated overexpression of constitutively active PKD resulted in enhanced AngII-induced aldosterone secretion; whereas overexpression of a dominant-negative PKD construct decreased AngII-stimulated aldosterone secretion. Thus, we demonstrate for the first time that PKD mediates acute AngII-induced aldosterone secretion.

Exogenous expression of caspase-14 induces tumor suppression in human salivary cancer cells by inhibiting tumor vascularization.

BACKGROUND: Current therapeutic approaches to salivary gland cancer are often associated with severe disfigurement and loss of glandular function, which are traumatic to the patients. Exploration of novel treatment approaches, such as gene therapy, is needed. MATERIALS AND METHODS: The human salivary gland cancer cell line HSG was transiently transfected with full length human caspase-14 cDNA. Photomicroscopy, BrdU assay, cell counting, MTT assay, and TUNEL assay were applied. To determine the tumorigenicity, tumor volume, tumor pathology and vascularization were analyzed in vivo. RESULTS: Cell growth and viability were inhibited significantly by transient caspase-14 expression. Caspase-14 expression resulted in a significant reduction of tumorigenicity. Importantly, a significant decrease in tumor blood vessel formation was observed. CONCLUSION: Salivary gland cancer cells underwent growth inhibition, cell death, and reduced tumorigenicity in vivo when exogenous caspase-14 was expressed, which could be due, in part, to an inhibitory effect of caspase-14 on tumor vascularization.