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The water and glycerol channel, aquaporin-3 (AQP3), plays an important role in the skin epidermis, with effects on hydration, permeability barrier repair and wound healing; therefore, information about the mechanisms regulating its expression is important for a complete understanding of skin function physiologically and in disease conditions. We previously demonstrated that histone deacetylase inhibitors (HDACi) induce the mRNA and protein expression of AQP3, in part through the p53 family, transcription factors for which acetylation is known to affect their regulatory activity. Another set of transcription factors previously shown to induce AQP3 expression and/or regulate skin function are the peroxisome proliferator-activated receptors (PPARs). Since there are reports that PPARs are also acetylated, we examined the involvement of these nuclear hormone receptors in HDACi-induced AQP3 expression. We first verified that a PPARγ agonist upregulated AQP3 mRNA and protein levels and that this increase was blocked by a PPARγ antagonist. We then showed that the PPARγ antagonist also inhibited AQP3 expression induced both by a broad-spectrum HDACi and an HDAC3-selective inhibitor. Interestingly, a PPARα antagonist also inhibited HDACi-induced AQP3 expression. These antagonist effects were observed in both primary mouse and normal human keratinocytes. Furthermore, PPARγ overexpression enhanced HDACi-stimulated AQP3 mRNA levels. Thus, our results suggest that PPARγ and/or PPARα may play a role in regulating AQP3 levels in the skin; based on the ability of PPAR agonists to promote epidermal differentiation and/or inhibit proliferation, topical PPAR agonists might be considered as a therapy for hyperproliferative skin disorders, such as psoriasis.

Our previous work shows that dioleoylphosphatidylglycerol (DOPG) accelerates corneal epithelial healing in vitro and in vivo by unknown mechanisms. Prior data demonstrate that DOPG inhibits toll-like receptor (TLR) activation and inflammation induced by microbial components (pathogen-associated molecular patterns, PAMPs) and by endogenous molecules upregulated in psoriatic skin, which act as danger-associated molecular patterns (DAMPs) to activate TLRs and promote inflammation. In the injured cornea, sterile inflammation can result from the release of the DAMP molecule, heat shock protein B4 (HSPB4), to contribute to delayed wound healing. Here, we show in vitro that DOPG inhibits TLR2 activation induced in response to HSPB4, as well as DAMPs that are elevated in diabetes, a disease that also slows corneal wound healing. Further, we show that the co-receptor, cluster of differentiation-14 (CD14), is necessary for PAMP/DAMP-induced activation of TLR2, as well as of TLR4. Finally, we simulated the high-glucose environment of diabetes to show that elevated glucose levels enhance TLR4 activation by a DAMP known to be upregulated in diabetes. Together, our results demonstrate the anti-inflammatory actions of DOPG and support further investigation into its development as a possible therapy for corneal injury, especially in diabetic patients at high risk of vision-threatening complications.

Prolonged inflammation and impaired re-epithelization are major contributing factors to chronic non-healing diabetic wounds; diabetes is also characterized by xerosis. Advanced glycation end products (AGEs), and the activation of toll-like receptors (TLRs), can trigger inflammatory responses. Aquaporin-3 (AQP3) plays essential roles in keratinocyte function and skin wound re-epithelialization/re-generation and hydration. Suberanilohydroxamic acid (SAHA), a histone deacetylase inhibitor, mimics the increased acetylation observed in diabetes. We investigated the effects of TLR2/TLR4 activators and AGEs on keratinocyte AQP3 expression in the presence and absence of SAHA. Primary mouse keratinocytes were treated with or without TLR2 agonist Pam3Cys-Ser-(Lys)4 (PAM), TLR4 agonist lipopolysaccharide (LPS), or AGEs, with or without SAHA. We found that (1) PAM and LPS significantly upregulated AQP3 protein basally (without SAHA) and PAM downregulated AQP3 protein with SAHA; and (2) AGEs (100 µg/mL) increased AQP3 protein expression basally and decreased AQP3 levels with SAHA. PAM and AGEs produced similar changes in AQP3 expression, suggesting a common pathway or potential crosstalk between TLR2 and AGEs signaling. Our findings suggest that TLR2 activation and AGEs may be beneficial for wound healing and skin hydration under normal conditions via AQP3 upregulation, but that these pathways are likely deleterious in diabetes chronically through decreased AQP3 expression.

Psoriasis is a common skin disorder characterized by hyperproliferation and aberrant differentiation of epidermal keratinocytes and inflammation. We previously demonstrated that phosphatidylglycerol (PG) can regulate keratinocyte function and suppress skin inflammation. Based on data suggesting that PG can inhibit toll-like receptor (TLR) activation induced by microorganisms and their components, we determined whether PG can inhibit TLR activation in response to anti-microbial peptides. These peptides, which are up-regulated in psoriasis, are known to function as danger-associated molecular patterns (DAMPs) to activate TLRs and the innate immune system. Since S100A9 is elevated in psoriatic skin and in animal models of psoriasis, we selected S100A9 as a representative anti-microbial peptide DAMP. In exciting results we show that in primary keratinocytes and a macrophage cell line PG suppressed inflammatory mediator production induced by recombinant S100A9 functioning through both TLR2 and TLR4. In addition, PG, but not phosphatidylcholine, inhibited downstream S100A9-elicited TLR2 and nuclear factor-κB activation. These results, to our knowledge previously unreported, demonstrate PG’s ability to inhibit DAMP-induced TLR activation, thereby reducing inflammatory signals. In addition, topical PG ameliorated skin lesions and inflammation in a mouse model of psoriasis. Together these results suggest the possibility of developing PG as a therapy for psoriasis.

Glycerol is used in many skin care products because it improves skin function. Anecdotal reports by patients on the National Psoriasis Foundation website also suggest that glycerol may be helpful for the treatment of psoriasis, although to date no experimental data confirm this idea. Glycerol entry into epidermal keratinocytes is facilitated by aquaglyceroporins like aquaporin-3 (AQP3), and its conversion to phosphatidylglycerol, a lipid messenger that promotes keratinocyte differentiation, requires the lipid-metabolizing enzyme phospholipase-D2 (PLD2). To evaluate whether glycerol inhibits inflammation and psoriasiform lesion development in the imiquimod (IMQ)-induced mouse model of psoriasis, glycerol’s effect on psoriasiform skin lesions was determined in IMQ-treated wild-type and PLD2 knockout mice, with glycerol provided either in drinking water or applied topically. Psoriasis area and severity index, ear thickness and ear biopsy weight, epidermal thickness, and inflammatory markers were quantified. Topical and oral glycerol ameliorated psoriasiform lesion development in wild-type mice. Topical glycerol appeared to act as an emollient to induce beneficial effects, since even in PLD2 knockout mice topical glycerol application improved skin lesions. In contrast, the beneficial effects of oral glycerol required PLD2, with no improvement in psoriasiform lesions observed in PLD2 knockout mice. Our findings suggest that the ability of oral glycerol to improve psoriasiform lesions requires its PLD2-mediated conversion to phosphatidylglycerol, consistent with our previous report that phosphatidylglycerol itself improves psoriasiform lesions in this model. Our data also support anecdotal evidence that glycerol can ameliorate psoriasis symptoms and therefore might be a useful therapy alone or in conjunction with other treatments.

Aldosterone is considered to be a link between hypertension and obesity; obese individuals have high serum levels of both sphingosine-1-phosphate (S1P) and very low-density lipoprotein (VLDL). S1P has been reported to be a novel stimulator of aldosterone secretion and phospholipase D (PLD) activity. VLDL has also been shown to stimulate aldosterone production in multiple zona glomerulosa cell models via PLD. PLD is an enzyme that hydrolyzes phosphatidylcholine to phosphatidic acid (PA) which can then be converted to diacylglycerol (DAG) by lipin-1. However, it is unclear which of the two lipid signals, PA or DAG, underlies PLD’s mediation of aldosterone production. We hypothesized that the S1P1 receptor (S1PR1) agonist, SEW2871, (and VLDL) induces steroidogenesis and therefore aldosterone production via lipin-1-mediated metabolism of PA to DAG, with our hypothesis focusing on DAG as the key lipid signal produced by PLD (indirectly). In HAC15 cells, lipin-1 was overexpressed using an adenovirus or inhibited using propranolol followed by treatment with or without SEW2871 (or VLDL) for 24 h. Steroidogenic gene expression and aldosterone levels were monitored by qRT-PCR and radioimmunoassay, respectively. We demonstrated that lipin-1 overexpression (OE) enhanced the SEW2871-stimulated 109-fold increase in CYP11B2 expression by 26% while lipin-1 inhibition decreased the SEW2871-stimulated 56-fold increase in CYP11B2 expression by 74%. While lipin-1 OE had no further effect, propranolol reduced SEW2871-stimulated increases in NR4A1 (2-fold) and NR4A2 (9-fold) mRNA levels by 22% and 52% respectively. The SEW2871-stimulated increase in aldosterone production was inhibited by propranolol (53%), although it was not enhanced by lipin-1 OE. Similar results were obtained with VLDL. Our results are, therefore, suggestive of DAG being the key lipid signal since regulating lipin-1 affects S1PR1 agonist- and VLDL-stimulated steroidogenic gene expression and ultimately, aldosterone production. Our study warrants further investigation into these steroidogenic signaling pathways which can lead to the identification of novel therapeutic targets such as lipin-1, or its downstream pathways, to potentially treat obesity-associated hypertension.

Indoleamine-2,3-dioxygenase (IDO) degrades the essential amino acid tryptophan resulting in tryptophan depletion and the accumulation of catabolites such as kynurenine. The expression/activity of IDO in various cells, including macrophages and dendritic cells, results in an inhibition of T-cell responses in a number of situations, such as toward allogeneic fetuses and tissue grafts. Psoriasis is an immune-mediated skin disease involving T cells; kynureninase and its generation of catabolites downstream of IDO are reported to play an important role in this disease. We hypothesized that mice lacking the IDO1 gene would exhibit a hyperactive immune response and an exacerbation of skin lesions in the imiquimod-induced mouse model of psoriasis. Littermate wild-type and IDO1-knockout mice were treated with imiquimod for 5 days, and the severity of psoriasiform skin lesions assessed using the psoriasis area and severity index (PASI), ear edema measured using a digital caliper, and thickness of the epidermis determined by histology. Expression of pro-inflammatory mediators and tryptophan-metabolizing enzymes was monitored using quantitative RT-PCR. Imiquimod increased ear edema, PASI scores, and epidermal thickness in both WT and IDO1 knockout mice; however, there were no differences observed between the 2 genotypes. There were also no differences in imiquimod’s induction of skin inflammatory mediators, indicating no effect of IDO1 gene loss in this psoriasis model. Although these data suggest a lack of involvement of IDO1 in psoriatic skin inflammation, other possible mechanisms, such as compensatory changes in other pathways and the involvement of the IDO2 isoform, must also be considered.

Skin serves not only as a protective barrier to microbial entry into the body but also as an immune organ. The outer layer, the epidermis, is composed predominantly of keratinocytes, which can be stimulated to produce proinflammatory mediators. Although some inflammation is useful to defend against infection, excessive or persistent inflammation can lead to the development of inflammatory skin diseases, such as psoriasis, a common skin disorder affecting approximately 2% of the US population. We have previously found that phosphatidylglycerol (PG) derived from soy can inhibit inflammation in a contact irritant ear edema mouse model. Here, we investigated the ability of soy PG to inhibit inflammatory mediator expression in response to activators of the pattern recognition receptors, toll-like receptor-2 (TLR2) and -4 (TLR4). We found that in epidermal keratinocytes, soy PG inhibited TLR2 and TLR4 activation and inflammatory mediator expression in response to a synthetic triacylated lipopeptide and lipopolysaccharide, respectively, as well as an endogenous danger-associated molecular pattern. However, at higher concentrations, soy PG alone enhanced the expression of some proinflammatory cytokines, suggesting a narrow therapeutic window for this lipid. Dioleoylphosphatidylglycerol (DOPG), but not dioleoylphosphatidylcholine, exerted a similar inhibitory effect, completely blocking keratinocyte inflammatory mediator expression induced by TLR2 and TLR4 activators as well as NFκB activation in a macrophage cell line (RAW264.7); however, DOPG was not itself proinflammatory even at high concentrations. Furthermore, DOPG had no effect on NFκB activation in response to a TLR7/8 agonist. Our results suggest that DOPG could be used to inhibit excessive skin inflammation. SIGNIFICANCE STATEMENT: Although inflammation is beneficial for clearing an infection, in some cases, the infection can be excessive and/or become chronic, thereby resulting in considerable tissue damage and pathological conditions. We show here that the phospholipid phosphatidylglycerol can inhibit the activation of toll-like receptors 2 and 4 of the innate immune system as well as the downstream inflammatory mediator expression in response to microbial component-mimicking agents in epidermal keratinocytes that form the physical barrier of the skin.

Osteoporosis, low bone mass that increases fracture susceptibility, affects approximately 75 million individuals in the United States, Europe and Japan, with the number of osteoporotic fractures expected to increase by more than three-fold over the next 50 years. Bone mass declines with age, although the mechanisms for this decrease are unclear. Aging enhances production of reactive oxygen species, which can affect bone formation and breakdown. The multiple sclerosis drug Tecfidera contains dimethylfumarate, which is rapidly metabolized to monomethylfumarate (MMF); MMF is thought to function through nuclear factor erythroid-derived-2-like-2 (NRF2), a transcription factor activated by oxidative stress which induces the expression of endogenous anti-oxidant systems. We hypothesized that MMF-elicited increases in anti-oxidants would inhibit osteopenia induced by ovariectomy, as a model of aging-related osteoporosis and high oxidative stress. We demonstrated that MMF activated NRF2 and induced anti-oxidant NRF2 target gene expression in bone marrow-derived mesenchymal stem cells. Sham-operated or ovariectomized adult female mice were fed chow with or without MMF and various parameters were monitored. Ovariectomy produced the expected effects, decreasing bone mineral density and increasing body weight, fat mass, bone marrow adiposity and serum receptor activator of nuclear factor-kappa-B ligand (RANKL) levels. MMF decreased fat but not lean mass. MMF improved trabecular bone microarchitecture after adjustment for body weight, although the unadjusted data showed few differences; MMF also tended to increase adjusted cortical bone and to reduce bone marrow adiposity and serum RANKL levels. Because these results suggest the possibility that MMF might be beneficial for bone, further investigation seems warranted.

We thank Lai and colleagues for their interest in our work and appreciate the opportunity to respond to their concerns. We agree with them that the mechanism by which monomethylfumarate acts is as yet unclear, which led us to investigate its effects on nuclear factor erythroid 2-related factor 2 (

The skin is the largest organ of the body, serving as an important barrier between the internal milieu and the external environment. The skin is also one of the first lines of defense against microbial infection and other hazards, and thus, the skin has important immune functions . This organ is composed of many cell types, including immune-active dendritic cells (epidermal Langerhans cells and dermal dendritic cells), connective tissue-generating dermal fibroblasts and pigment-producing melanocytes. Comprising the outer skin layer are the epidermal keratinocytes, the predominant cell of this layer, the epidermis , which provides both a mechanical barrier and a water -permeability barrier. Recent data suggest that aquaporins, a family of barrel-shaped proteins surrounding internal pores that allow the passage of water and, in some family members, small solutes such as glycerol , play critical roles in regulating various skin parameters. The involvement of different aquaporin family members in skin function is discussed.

Pioglitazone belongs to the class of drugs called thiazolidinediones (TZDs), which are widely used as insulin sensitizers in the treatment of diabetes. A major side effect of TZDs is fluid retention. The steroid hormone aldosterone also promotes sodium and fluid retention; however, the effect of pioglitazone on aldosterone production is controversial. We analyzed the effect of pioglitazone alone and in combination with angiotensin II (AngII) on the late rate-limiting step of adrenocortical steroidogenesis in human adrenocortical carcinoma HAC15 cells. Treatment with pioglitazone for 24hr significantly increased the expression of CYP11B2 and enhanced AngII-induced CYP11B2 expression. Despite the observed changes in mRNA levels, pioglitazone significantly inhibited AngII-induced aldosterone production and CYP11B2 protein levels. On the other hand, pioglitazone stimulated the expression of the unfolded protein response (UPR) marker DDIT3, with this effect occurring at early times and inhibitable by the PPARγ antagonist GW9962. The levels of DDIT3 (CHOP) and phospho-eIF2α (Ser51), a UPR-induced event that inhibits protein translation, were also increased. Thus, pioglitazone promotes CYP11B2 expression but nevertheless inhibits aldosterone production in AngII-treated HAC15 cells, likely by blocking global protein translation initiation through DDIT3 and phospho-eIF2α. In contrast, pioglitazone promoted AngII-induced CYP11B1 expression and cortisol production. Since cortisol enhances lipolysis, this result suggests the possibility that PPARs, activated by products of fatty acid oxidation, stimulate cortisol secretion to promote utilization of fatty acids during fasting. In turn, the ability of pioglitazone to stimulate cortisol production could potentially underlie the effects of this drug on fluid retention.

Aquaporin- (AQP) 3, a water and glycerol channel, plays an important role in epidermal function, with studies showing its involvement in keratinocyte proliferation, differentiation, and migration and in epidermal wound healing and barrier repair. Increasing speculation about the use of histone deacetylase (HDAC) inhibitors to treat skin diseases led us to investigate HDAC's role in the regulation of AQP3. The broad-spectrum HDAC inhibitor suberoylanilide hydroxamic acid induced AQP3 mRNA and protein expression in a dose- and time-dependent manner in normal keratinocytes. The SAHA-induced increase in AQP3 levels resulted in enhanced [3H]glycerol uptake in normal but not in AQP3-knockout keratinocytes, confirming that the expressed AQP3 was functional. Use of HDAC inhibitors with different specificities limited our exploration of the responsible HDAC member to HDAC1, HDAC2, or HDAC3. Cre-recombinase-mediated knockdown and overexpression of HDAC3 suggested a role for HDAC3 in suppressing AQP3 expression basally. Further investigation implicated p53 as a transcription factor involved in regulating HDAC inhibitor-induced AQP3 expression. Thus, our study supports the regulation of AQP3 expression by HDAC3 and p53. Because suberoylanilide hydroxamic acid is already approved to treat cutaneous T-cell lymphoma, it could potentially be used as a therapy for skin diseases like psoriasis, where AQP3 is abnormally expressed.

Lasp1 is an actin-binding, signaling pathway-regulated phosphoprotein that is overexpressed in several cancers. siRNA knockdown in cell lines retards cell migration, suggesting the possibility that Lasp1 upregulation influences cancer metastasis. Herein, we utilized a recently developed gene knockout model to assess the role of Lasp1 in modulating nontransformed cell functions. Wound healing and tumor initiation progressed more rapidly in Lasp1−/−mice compared with Lasp1+/+controls. Embryonic fibroblasts (MEFs) derived from Lasp1−/−mice also migrated more rapidly in vitro. These MEFs characteristically possessed increased focal adhesion numbers and displayed more rapid attachment compared with wild-type MEFs. Differential microarray analyses revealed alterations in message expression for proteins implicated in cell migration, adhesion, and cytoskeletal organization. Notably, the focal adhesion protein, lipoma preferred partner (LPP), a zyxin family member and putative Lasp1 binding protein, was increased about twofold. Because LPP gene disruption reduces cell migration, we hypothesize that LPP plays a role in enhancing the migratory capacity of Lasp1−/−MEFs, perhaps by modifying the subcellular localization of other motility-associated proteins. The striking contrast in the functional effects of loss of Lasp1 in innate cells compared with cell lines reveals distinct differences in mechanisms of motility and attachment in these models.

In order to understand the regulatory role of protein kinase C (PKC) in secretory epithelia, it is necessary to identify and characterize specific downstream targets. We previously identified one such protein in studies of gastric parietal cells. This protein was referred to as pp66 because it migrated with an apparent molecular mass of 66 kDa on SDS-polyacrylamide gels. The phosphorylation of pp66 is increased by the cholinergic agonist, carbachol, and by the PKC activator, phorbol-12-myristate-13-acetate, in a calcium-independent manner. In this study, we have purified pp66 to homogeneity and cloned the complete open reading frame. GenBankTM searches revealed a 45% homology with the Dictyostelium actin-binding protein, coronin, and approximately 67% homology with the previously cloned human and bovine coronin-like homologue, p57. pp66 appears to be most highly expressed in the gastrointestinal mucosa and in kidney and lung. Confocal microscopic studies of an enhanced green fluorescent protein fusion construct of pp66 in cultured parietal cells and in Madin-Darby canine kidney cells indicate that pp66 preferentially localizes in F-actin-rich regions. On the basis of our findings, we propose that pp66 may play an important, PKC-dependent role in regulating membrane/cytoskeletal rearrangements in epithelial cells. We have tentatively named this protein coroninse, because it appears to be highly expressed in secretory epithelia.

We have cloned five DNA fragments (-0.32, -0.48, -1.7, -3.2, and -5.1 kb) of the 5'-flanking region of the rat inducible nitric oxide synthase (iNOS) gene from rat genomic DNA. The functional importance of the 5'-flanking region was determined by transient expression of iNOS promoter-luciferase constructs in cultures of rat aortic smooth muscle cells. The -0.48 kb construct, containing one nuclear factor kappaB (NF-kappaB) binding site, expressed basal promoter activity but showed only a 1.5- and 1.7-fold increase in luciferase activity in response to lipopolysaccharide (LPS) or a cytokine mixture, respectively. However, the -3.2 kb construct (containing a second NF-kappaB binding site) showed full promoter activity with a 24-fold increase in response to LPS or cytokine mixture. The -5.1 kb construct showed no further increase in luciferase activity, suggesting that the 1.9 kb upstream of -3.2 kb may not be important in rat iNOS regulation. Rat iNOS promoter induction did not appear to be transcriptionally regulated by NO since NOS inhibitors did not affect induction. These data are in marked contrast to the mouse iNOS promoter in which a DNA sequence as short as a -85 bp, containing one NF-kappaB site, confers 10-fold inducibility by LPS. The present findings demonstrate that the rat iNOS gene is transcriptionally regulated by cytokines and LPS, but, unlike the mouse gene, the downstream NF-kappaB site does not appear to be a key region in responses to cytokines and LPS. These data suggest that the regulation of the rat gene may require the coexistence of at least two NF-kappaB sites or other elements upstream of -0.48 kb of the 5'-flanking region.

To explore the mechanism underlying the insulin-mimetic actions of vanadium and selenium we examined their effects on the mitogen activated protein/myelin basic protein kinases (MAPK) and ribosomal S6 protein kinases, which are among the best characterized of the kinases that comprise the phosphorylation cascade in insulin signal transduction. We observed a transient activation of MAPK and S6 kinases by insulin in rat adipocytes, while both sodium selenate and vanadyl sulphate produced prolonged activation of the kinases. Vanadyl sulphate stimulated the activity of MAPK and S6 kinase by as much as 6 fold and 15 fold, respectively. Pretreatment of the cells with genistein did not affect the activation of MAPK by insulin, but partially blocked the effects of sodium selenate and vanadyl sulphate. Genistein did not change the activation of S6 kinase by insulin, but blocked the activation in vanadyl sulphate- and sodium selenate-treated-cells, suggesting that a genistein sensitive tyrosine kinase may be involved in the activation by these two compounds. Rapamycin, a specific inhibitor of the p70s6k isoform of S6 kinase, partially reduced the activation of S6 kinase activity by sodium selenate, indicating a role for this kinase in the overall activity of the S6 kinase in sodium selenate-treated cells. A similar trend was noted in vanadyl sulphate-treated cells. Thus, this study supports the involvement of MAPK and S6 kinases in the insulin-mimetic actions of vanadium and selenium.

The mitogen-activated protein (MAP) kinases and ribosomal S6 protein kinases in the skeletal muscle of insulin-resistant long-term (2 and 6 months' duration) diabetic rats were investigated to understand further the changes in insulin intracellular signaling pathways that accompany diabetes. The effects of insulin-mimetic vanadium compounds on the activity of these kinases were also examined. In the insulin-resistant 2-month diabetic rats, the basal activities of MAP kinases were relatively unchanged, while the basal activities of S6 kinases were significantly increased. Intravenous injection of insulin moderately activated both the 42-kDa MAP kinase (p42mapk) and a 44-kDa MAP kinase (p44erk1) in the 2-month control rats but not in the 2-month diabetic rats. Insulin treatment markedly stimulated the activity of a novel 31-kDa S6 kinase and the previously described 90-kDa ribosomal S6 kinase encoded by one of the rsk genes (p90rsk) in the 2-month control rats, while the effect was substantially reduced in the diabetic rats. In the 6-month diabetic rats, the basal phosphotransferase activities of both MAP kinases were depressed threefold or greater. This correlated with reductions in the amount of immunoreactive p42mapk and p44erk1 proteins in extracts from the diabetic rats. The basal activity of the 31-kDa S6 kinase activity was also reduced fourfold in the 6-month diabetic rats. Treatment of the 2-month diabetic rats with vanadyl sulfate resulted in euglycemia, prevented the increase in the basal activity of S6 kinase, and improved the activation of S6 kinase by insulin. Treatment of the 6-month diabetic rats with an organic vanadium compound, bis(maltolato)oxovanadium(IV), also produced euglycemia and a restoration of the basal S6 kinase activities in these rats. The present findings indicate that insulin resistance associated with long-term diabetes may be linked with depressed signaling through these kinases and that this can be rectified by vanadium-containing compounds.

The predominant 40 S ribosomal protein S6 kinase in skeletal muscle extracts from insulin-treated rats was purified over 10,000-fold to near homogeneity with approximately 4.5% recovery of starting activity. This S6 kinase was resolved from the catalytic subunit of cAMP-dependent protein kinase only by the seventh and final column chromatography step. The purified S6 kinase migrated as a tight doublet of approximately 31 kDa on an SDS-polyacrylamide gel, and it was eluted from gel filtration columns with a similar apparent M(r), which indicated that the enzyme exists as a monomer. This S6 kinase was immunologically distinct from the other known insulin-activated S6 kinases, i.e. p70S6K and p90rsk. It was inhibited by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and beta-glycerophosphate at concentrations routinely used to stabilize p70S6K and p90rsk. In addition to S6, phosvitin was also a substrate, whereas myelin basic protein, casein, protamine, and histones were poorly phosphorylated if at all by the purified S6 kinase. The purified enzyme was inactivated upon incubation with serine/threonine-specific protein phosphatase 2A, which indicated that it may be an intermediary component in a cascade of insulin-activated protein kinases.

We monitored the activity of pulmonary microvascular endothelial-bound angiotensin-converting enzyme (ACE) in vivo by means of multiple indicator-dilution-type techniques, utilizing three different probes: the hydrolysis of two substrates, [3H]-benzoyl-Phe-Ala-Pro (BPAP) and [14C]benzoyl-Ala-Gly-Pro (BAGP), and the binding of the inhibitor [3H]RAC-X-65 (RAC), all measured during a single transpulmonary passage in anesthetized rabbits, placed on total heart bypass, so that both systemic and pulmonary circulations were fully supported by means of a two-channel extracorporeal pump. Experiments were performed at pulmonary blood flows (Qb) of 250, 400, 560, and 800 ml/min in control or indomethacin-pretreated rabbits. ACE activity was also compared to that of pulmonary microvascular endothelial-bound 5'-nucleotidase, by measuring the dephosphorylation of its natural substrate 5'-[14C]AMP. We calculated substrate utilization, mean lung transit time (t), and volume of distribution (i.e., central blood volume) of all substrates, as well as inhibitor binding. We also calculated Amax/Km and Bmax products of enzyme mass and kinetic constants for substrates and inhibitor, respectively. As Qb increased, Amax/Km values for all three substrates and Bmax increased linearly, indicating microvascular recruitment. In experiments in which either BPAP and 5'-AMP metabolism or BAGP metabolism and RAC binding were studied concomitantly, a linear relationship was observed between Qb-induced changes in Amax/Km values of BPAP vs 5'-AMP as well as in Amax/Km of BAGP vs Bmax of RAC. Similarly, increasing Qb increased central blood volume and decreased t. Indomethacin had no effect on most of the hemodynamic or enzyme parameters measured. We conclude that in vivo assays of ACE proceed as predicted by Michaelis-Menten kinetics and offer insights into pulmonary endothelial pathophysiology.

We investigated the early effects of radiation on pulmonary endothelial function in vivo 7-8 hr after exposure of rabbits to a single dose of 30 Gy to the chest. Utilizing multiple indicator-dilution techniques, we measured rates and kinetics of hydrolysis of the synthetic substrates [3H]benzoyl-Phe-Ala-Pro (BPAP) and [14C]benzoyl-Ala-Gly-Pro (BAGP) by endothelial-bound angiotensin-converting enzyme (ACE) and of 5′-[14C]-AMP by endothelial-bound 5′-nucleotidase (NCT) and binding of the synthetic ACE inhibitor [3H]RAC-X-65 during a single transpulmonary passage in anesthetized, artificially ventilated, open-chest rabbits in which both systemic and pulmonary circulations were fully supported by an extracorporeal pump. We have shown that these techniques and the use of the aforementioned probes provide reliable information on pulmonary endothelial function in vivo. Radiation to the chest produced endothelial ectoenzyme dysfunction, as reflected in altered available perfused capillary surface area and altered enzyme kinetics of all probes (decreases in substrate hydrolysis, inhibitor binding, first- and second-order kinetic constants) over a wide range of pulmonary blood flow values (reflecting approximately 60-200% of normal cardiac output). Indomethacin prevented most of these alterations in partially as well as fully recruited lungs. We conclude that impairment of endothelial ectoenzyme activity is an early event in the pathogenesis of radiation-induced lung damage, which occurs independently of hemodynamic influences and may involve synthesis of arachidonic acid metabolites.

Tumor protein D52 is expressed at relatively high levels in cells within the gastrointestinal tract that undergo classical exocytosis and is overexpressed in several cancers. Current evidence supports a role for D52 in the regulation of vesicular trafficking. D52 function(s) are regulated by calcium-dependent phosphorylation; however, the intracellular mechanisms that mediate this process are not well characterized. The goal of this study was to identify the calcium-dependent phosphorylation site(s) in D52 and to characterize the protein kinase(s) that mediate this phosphorylation. Using mass spectrometry and site-directed mutagenesis, we identified a single amino acid residue, S136, that undergoes increased phosphorylation upon elevation of intracellular Ca2+concentration. A phosphospecific antibody (pS136) was produced and used to characterize D52 kinase activity in gastric mucosal, colonic T84, and HEK293 cells. By using D52 as a substrate, a protein kinase with a molecular weight ( Mr) of ∼50 kDa was identified with “in gel” assays. This kinase comigrated with rat brain calcium/calmodulin-dependent protein kinase (CAMK2)α cross-reacted with pan-specific CAMK2 antibodies as well as with anti-active CAMK2 (pT286/287) antibody when activated. Carbachol-stimulated phosphorylation of S136was inhibited by the CAMK2 inhibitor KN93 (IC5038 μM) and by the calmodulin antagonist W7 (IC503.3 nM). A previously uncharacterized CAMK2 isoform, CAMK2δ6, which has the same domain structure and Mras CAM2α, was identified in gastric mucosa by RT-PCR. The cloned, expressed protein comigrated with D52 kinase and colocalized with D52 protein in T84 and HEK293 cells. These findings support a role for CAMK2δ6 in the mediation of D52 phosphorylation.

Lasp-1 (LIM and SH3 domain protein 1) is a multidomain actin-binding protein that is differentially expressed within epithelial tissues and brain. In the gastric mucosa, Lasp-1 is highly expressed in the HCl-secreting parietal cell, where it is prominently localized within the F-actin-rich subcellular regions. Histamine-induced elevation of parietal cell [cAMP]iincreases Lasp-1 phosphorylation, which is correlated with activation of HCl secretion. To determine whether Lasp-1 is involved in the regulation of HCl secretion in vivo, we generated a murine model with a targeted disruption of the Lasp-1 gene. Lasp-1-null mice had slightly lower body weights but developed normally and had no overt phenotypic abnormalities. Basal HCl secretion was unaffected by loss of Lasp-1, but histamine stimulation induced a more robust acid secretory response in Lasp-1-null mice compared with wild-type littermates. A similar effect of histamine was observed in isolated gastric glands on the basis of measurements of accumulation of the weak base [14C]aminopyrine. In addition, inhibition of the acid secretory response to histamine by H2 receptor blockade with ranitidine proceeded more slowly in glands from Lasp-1-null mice. These findings support the conclusion that Lasp-1 is involved in the regulation of parietal HCl secretion. We speculate that cAMP-dependent phosphorylation of Lasp-1 alters interactions with F-actin and/or endocytic proteins that interact with Lasp-1, thereby regulating the trafficking/activation of the H+, K+-ATPase (proton pump).

Huntingtin interacting protein 1 related (Hip1r) is an F-actin– and clathrin-binding protein involved in vesicular trafficking. In this study, we demonstrate that Hip1r is abundantly expressed in the gastric parietal cell, predominantly localizing with F-actin to canalicular membranes. Hip1r may provide a critical function in vivo, as demonstrated by extensive changes to parietal cells and the gastric epithelium in Hip1r-deficient mice. Electron microscopy revealed abnormal apical canalicular membranes and loss of tubulovesicles in mutant parietal cells, suggesting that Hip1r is necessary for the normal trafficking of these secretory membranes. Accordingly, acid secretory dynamics were altered in mutant parietal cells, with enhanced activation and acid trapping, as measured in isolated gastric glands. At the whole-organ level, gastric acidity was reduced in Hip1r-deficient mice, and the gastric mucosa was grossly transformed, with fewer parietal cells due to enhanced apoptotic cell death and glandular hypertrophy associated with cellular transformation. Hip1r-deficient mice had increased expression of the gastric growth factor gastrin, and mice mutant for both gastrin and Hip1r exhibited normalization of both proliferation and gland height. Taken together, these studies demonstrate that Hip1r plays a significant role in gastric physiology, mucosal architecture, and secretory membrane dynamics in parietal cells.

Heterozygous endothelial nitric oxide synthase (eNOS) deficiency is associated with normal endothelium‐dependent responses, however, little is known regarding the mechanisms that maintain or impair endothelial function with heterozygous eNOS deficiency. The goals of this study were to (1) determine mechanism(s) which serve to maintain normal endothelial function in the absence of a single eNOS gene; and (2) to determine whether heterozygous eNOS deficiency predisposes blood vessels to endothelial dysfunction in response to a high‐fat diet (HFD). Responses of carotid arteries were examined in wild‐type ( eNOS +/+ ) and heterozygous eNOS‐deficient ( eNOS +/− ) treated with either vehicle (saline), NG‐nitro‐L‐arginine (L‐NNA, 100 μmol/L), an inhibitor of nitric oxide synthase, or 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ, 1 μmol/L), an inhibitor of soluble guanylyl cyclase (sGC), and in eNOS +/+ and eNOS +/− mice fed a control (10%) or a 45% HFD (kcal from fat). Responses to acetylcholine (ACh) were similar in vehicle‐treated arteries from eNOS +/+ and eNOS +/− mice, and were equally inhibited by L‐NNA and ODQ. Phosphorylation of eNOS Ser1176, a site associated with increased eNOS activity, was significantly greater in eNOS +/− mice most likely as a compensatory response for the loss of a single eNOS gene. In contrast, responses to ACh were markedly impaired in carotid arteries from eNOS +/− , but not eNOS +/+ , mice fed a HFD. Vascular superoxide levels as well as plasma levels of the pro‐inflammatory cytokine interleukin‐6 (IL‐6) were selectively increased in HFD‐fed eNOS +/− mice. In reconstitution experiments, IL‐6 produced concentration‐dependent impairment of endothelial responses as well as greater increases in NADPH‐stimulated superoxide levels in arteries from eNOS +/− mice fed a control diet compared to eNOS +/+ mice. Our findings of increased Ser1176‐phosphorylation reveal a mechanism by which NOS‐ and sGC‐dependent endothelial function can be maintained with heterozygous eNOS deficiency. In addition, heterozygous eNOS deficiency predisposes blood vessels to developing endothelial dysfunction in response to a HFD. The impairment produced by a HFD in eNOS +/− mice appears to be mediated by IL‐6‐induced increases in vascular superoxide. These findings serve as an important example of eNOS haploinsufficiency, one that may contribute to the development of carotid artery disease in obese humans.

IQGAPs, GTPase-activating proteins with an IQ motif, are thought to regulate many actin cytoskeleton-based activities through interactions with Cdc42 and Rac. Recently, Cdc42 was implicated in regulation of gastric parietal cell HCl secretion, and IQGAP2 was immunolocalized with Cdc42 to F-actin-rich intracellular canalicular membranes of isolated gastric parietal cells in primary culture. Here we sought to define distribution and localization of IQGAP1 and IQGAP2 in major oxyntic (acid-secreting) gastric mucosal cell types and to determine whether secretory agonists modulate these proteins. Differential staining protocols were used to identify different cell populations (parietal, chief, surface/pit, and mucous neck cells) in semi-intact glands isolated from rabbit gastric mucosae and to characterize these same cells after dispersion and fractionation on isopycnic density gradients with simultaneous staining for F-actin, H+-K+-ATPase, and GSII lectin-binding sites. There was a pronounced increase in intracellular F-actin staining in dispersed chief cells, apparently from internalization of F-actin-rich apical membranes that normally abut the gland lumen. Therefore, other membrane-associated proteins might also be redistributed by disruption of cell-cell contacts. Western blot analyses were used to quantitate relative concentrations of IQGAPs in defined mucosal cell fractions, and gastric glands were used for in situ localizations. We detected uniform levels of IQGAP2 expression in oxyntic mucosal cells with predominant targeting to regions of cell-cell contact and nuclei of all cell types. IQGAP2 was not detected in parietal cell intracellular canaliculi. IQGAP1 expression was variable and targeted predominantly to the cortex of chief and mucous neck cells. Parietal cells expressed little or no IQGAP1 vs. other mucosal cell types. Phosphoprotein affinity chromatography, isoelectric focusing, and phosphorylation site analyses indicated that both IQGAP1 and IQGAP2 are phosphoproteins potentially regulated by [Ca2+]i/PKC and cAMP signaling pathways, respectively. Stimulation of glands with carbachol, which elevates [Ca2+]iand activates PKC, induced apparent translocation of IQGAP1, but not IQGAP2, to apical poles of chief (zymogen) and mucous neck cells. This response was mimicked by PMA but not by ionomycin or by elevation of [cAMP]iwith forskolin. Our observations support a novel, PKC-dependent role for IQGAP1 in regulated exocytosis and suggest that IQGAP2 may play a more general role in regulating cell-cell interactions and possibly migration within the gastric mucosa.

Lasp-1 has been identified as a signaling molecule that is phosphorylated upon elevation of [cAMP]i in pancreas, intestine and gastric mucosa and is selectively expressed in cells within epithelial tissues. In the gastric parietal cell, cAMP-dependent phosphorylation induces the partial translocation of lasp-1 to the apically directed F-actin-rich canalicular membrane, which is the site of active HCl secretion. Lasp-1 is an unusual modular protein that contains an N-terminal LIM domain, a C-terminal SH3 domain and two internal nebulin repeats. Domain-based analyses have recently categorized this protein as an epithelial representative of the nebulin family, which also includes the actin binding, muscle-specific proteins, nebulin, nebulette and N-RAP. In this study, we show that lasp-1 binds to non-muscle filamentous (F) actin in vitro in a phosphorylation-dependent manner. In addition, we provide evidence that lasp-1 is concentrated within focal complexes as well as in the leading edges of lamellipodia and the tips of filopodia in non-transformed gastric fibroblasts. In actin pull-down assays, the apparent K(d) of bacterially expressed his-tagged lasp-1 binding to F-actin was 2 micro M with a saturation stoichiometry of approximately 1:7. Phosphorylation of recombinant lasp-1 with recombinant PKA increased the K(d) and decreased the B(max) for lasp-1 binding to F-actin. Microsequencing and site-directed mutagenesis localized the major in vivo and in vitro PKA-dependent phosphorylation sites in rabbit lasp-1 to S(99) and S(146). BLAST searches confirmed that both sites are conserved in human and chicken homologues. Transfection of lasp-1 cDNA encoding for alanine substitutions at S(99) and S(146), into parietal cells appeared to suppress the cAMP-dependent translocation of lasp-1 to the intracellular canalicular region. In gastric fibroblasts, exposure to the protein kinase C activator, PMA, was correlated with the translocation of lasp-1 into newly formed F-actin-rich lamellipodial extensions and nascent focal complexes. Since lasp-1 does not appear to be phosphorylated by PKC, these data suggest that other mechanisms in addition to cAMP-dependent phosphorylation can mediate the translocation of lasp-1 to regions of dynamic actin turnover. The localization of lasp-1 to these subcellular regions under a range of experimental conditions and the phosphorylation-dependent regulation of this protein in F-actin rich epithelial cells suggests an integral and possibly cell-specific role in modulating cytoskeletal/membrane-based cellular activities.

Lasp-1 is a unique LIM and src homology 3 (SH3) domain-containing protein that was initially identified as a 40 kDa cAMP-dependent phosphoprotein in the HCl-secreting gastric parietal cell. Because cAMP is a potent stimulator of parietal cell acid secretion, we have hypothesized that changes in lasp-1 phosphorylation might be involved in the regulation of ion transport-related activities, perhaps by modulating interactions among cytoskeletal and/or vesicle-associated proteins. In this study, we demonstrate that the cAMP-dependent acid secretory agonist, histamine, induces a rapid, sustained rise in parietal cell lasp-1 phosphorylation and this increase in phosphorylation is closely correlated with the acid secretory response. In addition, elevation of intracellular cAMP concentrations appear to induce a partial redistribution of lasp-1 from the cell cortex, where it predominates along with the gamma-isoform of actin in unstimulated cells, to the beta-actin enriched, apically-directed intracellular canalicular region, which is the site of active proton transport in the parietal cell. Additional studies demonstrate that although lasp-1 mRNA and protein are expressed in a wide range of tissues, the expression is specific for certain actin-rich cell types present within these tissues. For example, gastric chief cells, which contain relatively little F-actin and secrete the enzyme, pepsinogen, by regulated exocytosis, do not appear to express lasp-1. Similarly, lasp-1 was not detected in pancreatic acinar cells, which secrete enzymes by similar mechanisms and also contain relatively low levels of F-actin. Lasp-1 also was not detectable in proximal tubules in the kidney, in gastrointestinal smooth muscle, heart or skeletal muscle. In contrast, expression was prominent in the cortical regions of ion-transporting duct cells in the pancreas and in the salivary parotid gland as well as in certain F-actin-rich cells in the distal tubule/collecting duct. Interestingly, moderate levels of expression were also detected in podocytes present in renal glomeruli and in vascular endothelium. In primary cultures of gastric fibroblasts, lasp-1 was present mainly within the tips of lamellipodia and at the leading edges of membrane ruffles. Taken together these results support the hypothesis that the lasp-1 plays an important role in the regulation of dynamic actin-based, cytoskeletal activities. Agonist-dependent changes in lasp-1 phosphorylation may also serve to regulate actin-associated ion transport activities, not only in the parietal cell but also in certain other F-actin-rich secretory epithelial cell types.

Objective: The objectives of the present study were to determine functional (i.e., heparin-releasable) and intracellular (i.e., heparin-non-releasable) cardiac lipoprotein lipase (LPL) activity during the development of hypertension in spontaneously hypertensive (SHR) rats. Methods : Male WKY and SHR rats were killed before (7–8 weeks of age) and following (15–16 weeks of age) the development of severe hypertension in SHR rats. LPL activity in coronary perfusates was determined by retrogradely perfusing the hearts with heparin (5 U/ml). Cardiac myocytes were also isolated from the two groups of rats by collagenase digestion, and surface-bound and intracellular LPL activity measured. Results : With the development of hypertension in SHR rats, there was a concomitant and progressive reduction in the heparin-releasable coronary endothelial LPL activity. Neither insulin action nor cell-associated enzyme activity could explain this low LPL activity in coronary blood vessels. However, acute vasodilation with nifedipine (a Ca2+ influx blocker) or CGS-21680 (A2-purinergic receptor agonist) increased the peak heparin-releasable LPL activity in hearts isolated from SHR rats. Conclusions : Our results indicate that hypertension per se may play a significant role in regulating cardiac LPL activity, and hence fatty acid supply to the hypertensive SHR rat heart.

We examined the distribution of mitogen-activated protein (MAP) kinase, S6 kinase, and casein kinase II (CK-II) in the muscle, spleen, brain, and testes of Wistar rats. It was observed that spleen extracts contained the highest activity of all the kinases. Anion-exchange chromatography of spleen extracts by a MonoQ column resolved a single peak of myelin basic protein phosphotransferase activity that eluted after the usual position of the previously described p42 and p44 MAP kinases. Immunoblotting of the peak fractions with anti-MAP kinase antibody did not detect any immunoreactive bands that coincided with the activity peak, suggesting that the activity may represent a potentially novel MAP kinase. The MonoQ fractionation also resolved a single peak of phosvitin phosphotransferase activity which coincided with the intensity of two immunoreactive bands of 39 and 43 kilodaltons that were detected with antibodies against CK-II. The chromatographic behaviour and immunoblotting data indicate that the phosvitin kinase peak represented CK-II and suggested that the rat spleen CK-II had a molecular structure of αα′β2. Furthermore, using an intact rat model, we showed that the potentially novel spleen MAP kinase and CK-II were markedly activated following intravenous injection of insulin. The significance of these findings remains to be determined.Key words: mitogen-activated protein kinase, S6 kinase, casein kinase II, insulin, spleen.

With the advent of thrombolytic therapy for acute ischemic stroke it may soon be possible to rapidly restore blood flow to an ischemic area of the brain. However, reperfusion may carry its own attendant risk of further tissue injury. There is an increasing body of evidence which suggests that neutrophils play an important role in mediating tissue damage during acute ischemic stroke [1, 2, 4, 5]. Moreover, there are suggestions that neutrophils may be at least partially responsible for the “no reflow” phenomenon [3].