Involvement of p38 MAP kinase and Smad3 in TGF-beta-mediated mast cell functions.

Transforming growth factor-beta (TGF-beta) modulates functions of bone marrow-derived cultured mast cells (BMMCs); cell maturation (up-regulation of mouse mast cell proteases (mmcps)), growth arrest and migration. We investigated the roles of p38 MAP kinase and Smad3 in TGF-beta-mediated cell responses in BMMCs. Treating BMMCs with TGF-beta induced the phosphorylation of p38 within 2 h and persisted for 24 h. The involvement of p38 in TGF-beta-induced cell responses depended upon mast cell functions; it was necessary for up-regulation of mmcp-1 and migration, but not for up-regulation of mmcp-7 and inhibition of metabolic activity. New protein synthesis was required for the up-regulation of mmcp-1 but not mmcp-7 in response to TGF-beta treatment, and stabilization of mRNA was partially responsible for the increase in gene transcript of mmcp-1. The decrease in metabolic activity in response to TGF-beta treatment was smaller in Smad3-deficient BMMCs compared to wild-type BMMCs. Maximal migration was detected at a TGF-beta concentration of 40 fM in wild-type BMMCs, whereas TGF-beta-induced migration was absent in Smad3-deficient BMMCs. Thus, the roles of p38 and Smad3 are different among TGF-beta-mediated cell responses in BMMCs.

Transcriptional regulation of mouse mast cell protease-7 by TGF-beta.

Mouse mast cell protease-7 (mmcp-7) is a tryptase predominantly expressed in differentiated connective tissue-type mast cells. Previous study revealed that transforming growth factor-beta (TGF-beta) increases gene transcript of mmcp-7 in mast cells. The present study explored molecular mechanism of the up-regulation of mmcp-7 by TGF-beta. Luciferase-based reporter assays using deletion and point mutations of mmcp-7 promoter showed a critical region spanning nt -126 to -122 relative to the transcriptional start site, a Smad-binding element, for transcriptional activation by the TGF-beta pathway. In addition, a region from nt -104 to -98, a TPA-responsive element, was also necessary for the transactivation. Consistent with the current model for the TGF-beta signaling, Smad4 was required for the transcription of mmcp-7 by Smad3, a signal mediator of TGF-beta. Treatment with TGF-beta in mast cells resulted in the differential gene induction of the AP-1 components, i.e., transient induction of c-fos but not of c-jun and junB. Expression of c-fos further enhanced Smad3 and Smad4-induced transcription of mmcp-7, whereas c-jun expression inhibited the transcription. Our results suggest that TGF-beta stimulates mmcp-7 transcription through the Smad3-Smad4 pathway as well as c-fos induction, and that the AP-1 components distinctly related with the TGF-beta pathway.

Evaluation of meat meal, chicken meal, and corn gluten meal as dietary sources of protein in dry cat food.

The nutritional value of meat meal (MM), chicken meal (CM), and corn gluten meal (CGM) as dietary sources of protein in dry food formulated for adult cats was evaluated. Twelve healthy adult cats (11 males and 1 female) were used. Dry diets containing MM, CM, or CGM as the main protein source were given for a 3-week period in a 3 x 3 Latin-square design. Digestion and balance experiments were conducted during the last 7 d of each period. In addition, freshly voided urine was taken to determine urinary pH and number of struvite crystals. As compared with the CM diet, dry-matter digestibility was higher and lower for the MM and CGM groups, respectively. Percentages of nitrogen (N) absorption and N retention to N intake were higher in the MM group, and N utilization was not different between the CM group and the CGM group. All cats excreted alkaline urine (pH > 7). Urinary pH, struvite activity product, and number of struvite crystals in urine were lower for the CGM group. There was no difference in retention of calcium and magnesium among the groups. From the point of view of digestibility and N utilization, MM is superior to CGM, and CM is better than or equivalent to CGM as a protein source of dry foods for adult cats. However, when CM is used as a dietary protein source, some manipulation of dietary base excess may be needed to control urinary acid-base balance, because CM contains higher calcium and phosphorus.

Up-regulation of mouse mast cell protease-6 gene by transforming growth factor-beta and activin in mast cell progenitors.

Previous studies have revealed that members of the transforming growth factor-beta (TGF-beta) including TGF-beta1 and activin A modulate the function of mast cells. Here we show the up-regulation of mouse mast cell protease-6 (mMCP-6), which is expressed in differentiated mast cells, by TGF-beta1 and activin A in bone marrow-derived cultured mast cell progenitors (BMCMCs). Quantitative real time RT-PCR analyses revealed that the mRNA level of mMCP-6 was slightly but reproducibly increased by treatment with TGF-beta1 or activin A, which was regulated at the transcription level. Reporter assays showed that Smad3, a signal mediator of the TGF-beta/activin pathway, was responsible for the transcription. The TGF-beta response element is located at -153 bp relative to the transcription initiation site, CAGA. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, the heart and skeletal muscle, also stimulated the transcription of mMCP-6. The region at -166 bp, GACCTG, was responsible for MITF-induced transcription. Mutations of the CAGA motif and the MITF responsive site indicated that the MITF site of mMCP-6 promoter is indispensable for the transcriptional activation by a constitutively active TGF-beta receptor (ALK5-TD), whereas the CAGA motif is dispensable for transcription by MITF. Transcriptional activation of mMCP-6 by the TGF-beta pathway was differently interacted with that by MITF isoform; ALK5-TD further enhanced MITF-E-induced transcription, whereas MITF-M-induced transcription abolished responsiveness to ALK5-TD. The positive regulation of mMCP-6 by the TGF-beta/activin pathway and the differential regulation by the MITF isoform suggest a rigorous regulation of mast cell function as effector cells of immune response.

Evaluation of effects of dietary carbohydrate on formation of struvite crystals in urine and macromineral balance in clinically normal cats.

OBJECTIVE: To evaluate effects of dietary carbohydrate on urine volume; struvite crystal formation; and calcium, phosphorus, and magnesium balance in clinically normal cats. ANIMALS: 21 healthy adult cats (15 sexually intact males and 6 sexually intact females). PROCEDURE: Diets containing no carbohydrate source (control diet), control plus starch, or control plus fiber were given in a 3 X 3 Latin-square design. The diets were available ad libitum in study 1 (n = 12) and given under restrictions in study 2 (9) to equalize daily intakes of crude protein among the 3 groups. Formation of struvite crystals and balance of calcium, phosphorus, and magnesium were measured. RESULTS: Urine volume was lower in the starch group and fiber group in study 1, whereas no differences were detected among the groups in study 2. Urinary pH and struvite activity product were higher in the starch group in both studies, and the fiber group also had higher struvite activity product in study 2. In both studies, urinary concentrations of HCl-insoluble sediment were higher in the starch group and fiber group. In the fiber group, a net loss of body calcium, phosphorus, and magnesium was detected in study 2. CONCLUSIONS AND CLINICAL RELEVANCE: Starch and fiber in diets potentially stimulate formation of struvite crystals. Hence, reducing dietary carbohydrate is desirable to prevent struvite urolith formation. In addition, a net loss of body calcium, phosphorus, and magnesium during feeding of the fiber diet suggests that dietary inclusion of insoluble fiber could increase macromineral requirements of cats.

Transcriptional activation of mouse mast cell Protease-7 by activin and transforming growth factor-beta is inhibited by microphthalmia-associated transcription factor.

Previous studies have revealed that activin A and transforming growth factor-beta1 (TGF-beta1) induced migration and morphological changes toward differentiation in bone marrow-derived cultured mast cell progenitors (BMCMCs). Here we show up-regulation of mouse mast cell protease-7 (mMCP-7), which is expressed in differentiated mast cells, by activin A and TGF-beta1 in BMCMCs, and the molecular mechanism of the gene induction of mmcp-7. Smad3, a signal mediator of the activin/TGF-beta pathway, transcriptionally activated mmcp-7. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, and heart and skeletal muscle, inhibited Smad3-mediated mmcp-7 transcription. MITF associated with Smad3, and the C terminus of MITF and the MH1 and linker region of Smad3 were required for this association. Complex formation between Smad3 and MITF was neither necessary nor sufficient for the inhibition of Smad3 signaling by MITF. MITF inhibited the transcriptional activation induced by the MH2 domain of Smad3. In addition, MITF-truncated N-terminal amino acids could associate with Smad3 but did not inhibit Smad3-mediated transcription. The level of Smad3 was decreased by co-expression of MITF but not of dominant-negative MITF, which resulted from proteasomal protein degradation. The changes in the level of Smad3 protein were paralleled by those in Smad3-mediated signaling activity. These findings suggest that MITF negatively regulates Smad-dependent activin/TGF-beta signaling in a tissue-specific manner.

Degranulation in RBL-2H3 cells: regulation by calmodulin pathway.

Involvement of the calmodulin pathway in Ca2+-induced degranulation was evaluated in RBL-2H3 mast cells. Pretreatment of RBL-2H3 cells with a calmodulin antagonist, W-13, blocked ionomycin-dependent release of beta-hexosaminidase into the supernatant, although W-13 treatment alone slightly but significantly increased the release. Ca2+/calmodulin activates various protein kinases and phosphatases including myosin-light chain kinase (MLCK), calmodulin-dependent protein kinases (CaMKs), and calcineurin. When RBL-2H3 cells were pretreated with a MLCK inhibitor, ML-7, or a CaMKs inhibitor, KN-93, the ionomycin-dependent release of beta-hexosaminidase into the supernatant was inhibited. In addition, pretreatment with calcineurin inhibitors, cyclosporin A and FR901725, resulted in blockage of the ionomycin-dependent release of beta-hexosaminidase into the supernatant. Our results indicate that Ca2+/calmodulin, activated calmodulin, is indispensable for Ca2+-induced degranulation, and that within the calmodulin pathways, at least MLCK, CaMKs and calcineurin positively regulate the release of granules initiated by increasing cytosolic Ca2+ concentrations in RBL-2H3 cells.

Effects of a high-protein diet versus dietary supplementation with ammonium chloride on struvite crystal formation in urine of clinically normal cats.

OBJECTIVE: To evaluate the effects of a high-protein diet versus dietary supplementation with ammonium chloride (NH4Cl) on struvite crystal formation in the urine of clinically normal cats by measuring the urine concentration of hydrochloric acid (HCl)-insoluble sediment, urine pH, struvite activity product (SAP), number of struvite crystals in urine, and urine volume. ANIMALS: 23 healthy adult cats. PROCEDURE: Urine was fractionated by centrifugation with subsequent extraction of the sediment with 1 N HCl (study 1). Diets containing either 29% crude protein or 55% crude protein were fed to cats in a crossover trial of 3 weeks/period (study 2). Diets supplemented with either sodium chloride (NaCl) or NH4Cl were fed, by use of a 3 x 3 Latin-square design with 3 wk/period (study 3). In studies 2 and 3, urine samples were collected for the last 7 days of each period. RESULTS: The HCl-insoluble sediment contained Tamm-Horsfall glycoprotein (THP; study 1). The high-protein diet (study 2) and dietary supplementation with NH4Cl (study 3) resulted in a decrease in urine pH, SAP, and the number of struvite crystals in urine. However, the high-protein diet decreased urine concentrations of HCl-insoluble sediment containing THP (study 2), in contrast to the NH4Cl supplementation that increased urine volume without a significant effect on the urine concentration of the HCl-insoluble sediment (study 3). CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that compared with dietary supplementation with NH4Cl, the high-protein diet is preferable as a urine acidifier for the prevention of struvite crystal formation in clinically normal cats.

Role of activin A in murine mast cells: modulation of cell growth, differentiation, and migration.

Activins, members of the transforming growth factor-beta (TGF-beta) superfamily, are potent growth and differentiation factors. Our previous studies revealed that activin A, a homodimer of inhibin/activin beta(A), was induced in mast cells and peritoneal macrophages in response to their activation. In the present study, we examined the roles of activin A in murine bone marrow-derived, cultured mast cell progenitors (BMCMCs), which expressed gene transcripts for molecules involved in activin signaling, suggesting that BMCMCs could be target cells of activin A. Treatment of activin A inhibited 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide uptake into BMCMCs in a dose-dependent manner. The IC(50) concentration was 2.1 nM, which was less potent than 185 pM TGF-beta(1). Activin A treatment caused morphological changes toward the differentiated cells at 2 nM and up-regulated mRNA of mouse mast cell protease-1 (mMCP-1), a marker enzyme of mature mucosal mast cells, at 1 nM. Activin A also showed activity in inducing migration of BMCMCs; the optimal concentration for maximal migration was 10 pM, which was much lower than the concentrations to inhibit cell growth and to activate the mMCP-1 gene. Taking the present results together with our previous results, it is suggested that activin A secreted from activated immune cells recruits mast cell progenitors to sites of inflammation and that with increasing activin A concentration, the progenitors differentiate into mature mast cells. Thus, activin A may positively regulate the functions of mast cells as effector cells of the immune system.

Calcium-regulated expression of activin A in RBL-2H3 mast cells.

The present study examined the regulatory expression of activin A, a potent growth and differentiation factor, in rat basophilic leukemia (RBL-2H3) mast cells. Treatment of RBL-2H3 cells sensitized with anti-dinitrophenyl IgE with multivalent dinitrophenyl led to a clear increase in RT-PCR products of inhibin/activin beta(A). The steady-state mRNA of inhibin/activin beta(A) was also induced by increasing cytosolic Ca(2+) concentration with ionomycin, which required de novo protein synthesis, and was regulated at the transcriptional level. Pretreatment of RBL-2H3 cells with antagonists or inhibitors for the calmodulin pathway blocked ionomycin-dependent inhibin/activin beta(A) transcription and mRNA induction, suggesting the involvement of calmodulin-dependent kinase (CaMK) and calcineurin. The ionomycin-dependent inhibin/activin beta(A) induction was also partially blocked by preincubation with c-Jun NH(2)-terminal kinase (JNK) and p38 kinase inhibitors, but not with MEK1 inhibitor. These results suggest that inhibin/activin beta(A) gene activation is achieved by the JNK and p38 kinase activation through the calmodulin pathway in mast cells.

Comparison of corn gluten meal and meat meal as a protein source in dry foods formulated for cats.

OBJECTIVE: To compare the nutritional value of corn gluten meal (CGM) and meat meal (MM) as a dietary source of protein in dry food formulated for adult cats. ANIMALS: 8 healthy adult cats (4 males and 4 females). PROCEDURE: Diets containing CGM or MM as the main protein source were each fed for a 3-week period in a crossover study. Digestibility and nutritional balance experiments were conducted during the last 7 days of each period. Furthermore, freshly voided urine was obtained to measure urinary pH, struvite crystals, and sediment concentrations. RESULTS: Daily food intake and dry-matter digestibility were significantly higher for the MM diet. Fecal moisture content also was higher for the MM diet. Apparent nitrogen (N) absorption and N retention were higher for the MM diet, even when values were expressed as a percentage to account for differences in N intake. Urinary pH, struvite activity product, number of struvite crystals in urine, and urinary sediment concentrations were not different between diets. Retention of calcium, phosphorus, and magnesium was lower for the CGM diet, and cats lost body calcium and magnesium when fed the CGM diet. CONCLUSIONS AND CLINICAL RELEVANCE: Meat meal was superior to CGM as a protein source in dry foods formulated for cats, because dry-matter digestibility and N utilization were higher for the MM diet. In addition, net loss of body calcium and magnesium for the CGM diet suggests that mineral requirements increase when CGM is used as a protein source.