Cleavage of huntingtin by apopain, a proapoptotic cysteine protease, is modulated by the polyglutamine tract.

Apoptosis has recently been recognized as a mode of cell death in Huntington disease (HD). Apopain, a human counterpart of the nematode cysteine protease death-gene product, CED-3, has a key role in proteolytic events leading to apoptosis. Here we show that apoptotic extracts and apopain itself specifically cleave the HD gene product, huntingtin. The rate of cleavage increases with the length of the huntingtin polyglutamine tract, providing an explanation for the gain-of-function associated with CAG expansion. Our results show that huntingtin is cleaved by cysteine proteases and suggest that HD might be a disorder of inappropriate apoptosis.

HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain.

Huntington disease (HD) is associated with the expansion of a polyglutamine tract, greater than 35 repeats, in the HD gene product, huntingtin. Here we describe a novel huntingtin interacting protein, HIP1, which co-localizes with huntingtin and shares sequence homology and biochemical characteristics with Sla2p, a protein essential for function of the cytoskeleton in Saccharomyces cerevisiae. The huntingtin-HIP1 interaction is restricted to the brain and is inversely correlated to the polyglutamine length in huntingtin. This provides the first molecular link between huntingtin and the neuronal cytoskeleton and suggests that, in HD, loss of normal huntingtin-HIP1 interaction may contribute to a defect in membrane-cytoskeletal integrity in the brain.

PRDM14 promotes malignant phenotype and correlates with poor prognosis in colorectal cancer.

BACKGROUND: Emerging evidence suggests that stemness in cancer cells is a cause of drug resistance or metastasis and is an important therapeutic target. PR [positive regulatory domain I-binding factor 1 (PRDI-BF1) and retinoblastoma protein-interacting zinc finger gene (RIZ1)] domain containing 14 (PRDM14), that regulates pluripotency in primordial germ cell, has reported the overexpression and function of stemness in various malignancies, suggesting it as the possible therapeutic target. However, to our knowledge, there have been no reports on the expression and function of PRDM14 in colorectal cancer (CRC). Therefore, we investigated the expression and the role of PRDM14 in CRC. METHODS: We performed immunohistochemistry evaluations and assessed PRDM14 expression on 414 primary CRC specimens. Colon cancer cell lines were subjected to functional and stemness assays in vitro and in vivo. RESULTS: We found that PRDM14 positive staining exhibited heterogeneity in the CRC primary tumor, especially at the tumor invasion front. The aberrant expression of PRDM14 at the invasion front was associated with lymph node metastasis and disease stage in patients with CRC. Furthermore, the multivariate analysis revealed high PRDM14 expression as an independent prognostic factor in the patients with Stage III CRC. Overexpression of PRDM14 enhanced the invasive, drug-resistant and stem-like properties in colon cancer cells in vitro and tumorigenicity in vivo. CONCLUSION: Our findings suggest that PRDM14 is involved in progression and chemoresistance of CRC, and is a potential prognostic biomarker and therapeutic target in the CRC patients.

Mechanistic interpretation of rift valley formation.

The stress distribution on the lithospheric plate due to excess magma pressure is obtained from an exact solution of the three-dimensional theory of elasticity. The analysis indicates that rift valley formation and associated structural and geophysical characteristics can be suitably explained by dike-like intrusions of magma or igneous mush into the lithospheric plate.

Cow's milk increases the activities of human nuclear receptors peroxisome proliferator-activated receptors alpha and delta and retinoid X receptor alpha involved in the regulation of energy homeostasis, obesity, and inflammation.

The nuclear peroxisome proliferator-activated receptors (PPAR) have been shown to play crucial roles in regulating energy homeostasis including lipid and carbohydrate metabolism, inflammatory responses, and cell proliferation, differentiation, and survival. Because PPAR agonists have the potential to prevent or ameliorate diseases such as hyperlipidemia, diabetes, atherosclerosis, and obesity, we have explored new natural agonists for PPAR. For this purpose, cow's milk was tested for agonistic activity toward human PPAR subtypes using a reporter gene assay. Milk increased human PPARalpha activity in a dose-dependent manner with a 3.2-fold increase at 0.5% (vol/vol). It also enhanced human PPARdelta activity in a dose-dependent manner with an 11.5-fold increase at 0.5%. However, it only slightly affected human PPARgamma activity. Ice cream, butter, and yogurt also increased the activities of PPARalpha and PPARdelta, whereas vegetable cream affected activity of PPARdelta but not PPARalpha. Skim milk enhanced the activity of PPAR to a lesser degree than regular milk. Milk and fresh cream increased the activity of human retinoid X receptor (RXR)alpha as well as PPARalpha and PPARdelta, whereas neither affected vitamin D3 receptor, estrogen receptors alpha and beta, or thyroid receptors alpha and beta. Both milk and fresh cream were shown by quantitative real-time PCR to increase the quantity of mRNA for uncoupling protein 2 (UCP2), an energy expenditure gene, in a dose-dependent manner. The increase in UCP2 mRNA was found to be reduced by treatment with PPARdelta-short interfering (si)RNA. This study unambiguously clarified at the cellular level that cow's milk increased the activities of human PPARalpha, PPARdelta, and RXRalpha. The possible role in enhancing the activities of PPARalpha, PPARdelta, and RXRalpha, and the health benefits of cow's milk were discussed.

Additional renoprotective effects of azelnidipine combined with angiotensin receptor blockers in patients with diabetic nephropathy.

Azelnidipine has been reported to have antioxidant effects and attenuates tubulointerstitial ischemia. The aim of the present study was to determine whether azelnidipine exerts additional renoprotective effects to angiotensin II receptor blockers (ARBs) in hypertensive patients with diabetic nephropathy and microalbuminuria. 45 hypertensive patients with diabetes mellitus and microalbuminuria who were already being treated with ARBs were enrolled in this study. Azelnidipine was added to the drug treatment of 30 patients (8 mg/day, n = 15, or 16 mg/day, n = 15) whilst the remaining 15 control patients were not treated with azelnidipine. In all patients, urinary 8-hydroxydeoxyguanosine (8-OHdG) levels and urinary liver-type fatty acid-binding protein (L-FABP) levels were significantly correlated (r = 0.587, p = 0.0006). However, urinary albumin excretion (UAE) was not correlated with the levels of urinary 8-OHdG (r = 0.1975, p = 0.2956) or urinary L-FABP (r = 0.2057, p = 0.2759). Azelnidipine significantly reduced UAE, urinary 8-OHdG and urinary L-FABP after 6 (p < 0.05) and 12 months (p < 0.05). Although blood pressure was comparable between the azelnidipine doses of 8 and 16 mg/day, the UAE (p < 0.05 after 12 months), urinary 8-OHdG (p < 0.05 after 6 and 12 months) and urinary L-FABP (p < 0.05 after 6 and 12 months) levels were more significantly reduced in patients receiving the higher dose of 16 mg/day. These data may suggest that the addition of azelnidipine treatment to therapy with ARBs has dose-dependent antioxidant and renoprotective effects beyond blood pressure-lowering effects in hypertensive diabetic nephropathy patients.

Model-based drug development.

The low productivity and escalating costs of drug development have been well documented over the past several years. Less than 10% of new compounds that enter clinical trials ultimately make it to the market, and many more fail in the preclinical stages of development. These challenges in the "critical path" of drug development are discussed in a 2004 publication by the US Food and Drug Administration. The document emphasizes new tools and various opportunities to improve drug development. One of the opportunities recommended is the application of "model-based drug development (MBDD)." This paper discusses what constitutes the key elements of MBDD and how these elements should fit together to inform drug development strategy and decision-making.

Involvement of Ras and Ral in chemotactic migration of skeletal myoblasts.

In skeletal myoblasts, Ras has been considered to be a strong inhibitor of myogenesis. Here, we demonstrate that Ras is involved also in the chemotactic response of skeletal myoblasts. Expression of a dominant-negative mutant of Ras inhibited chemotaxis of C2C12 myoblasts in response to basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-1), key regulators of limb muscle development and skeletal muscle regeneration. A dominant-negative Ral also decreased chemotactic migration by these growth factors, while inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase (MEK) showed no effect. Activation of the Ras-Ral pathway by expression of an activated mutant of either Ras, the guanine-nucleotide dissociation stimulator for Ral, or Ral resulted in increased motility of myoblasts. The ability of Ral to stimulate motility was reduced by introduction of a mutation which prevents binding to Ral-binding protein 1 or phospholipase D. These results suggest that the Ras-Ral pathway is essential for the migration of myoblasts. Furthermore, we found that Ras and Ral are activated in C2C12 cells by bFGF, HGF and IGF-1 and that the Ral activation is regulated by the Ras- and the intracellular Ca(2+)-mediated pathways. Taken together, our data indicate that Ras and Ral regulate the chemotactic migration of skeletal muscle progenitors.

Urinary liver-type fatty acid-binding protein levels for differential diagnosis of idiopathic focal glomerulosclerosis and minor glomerular abnormalities and effect of low-density lipoprotein apheresis.

AIMS: Focal glomerulosclerosis (FGS) and minor glomerular abnormalities are kidney diseases characterized by massive proteinuria. Urinary liver-type fatty acid-binding protein (L-FABP), an intracellular carrier protein of free fatty acids, is expressed in proximal tubules of the human kidney. Patients with FGS show significant improvement with low-density lipoprotein (LDL) apheresis. The aim of the present study was to determine whether urinary L-FABP levels differ between patients with FGS and those with minor glomerular abnormalities and whether levels are altered by LDL apheresis. PATIENTS AND METHODS: There were 24 patients with minor glomerular abnormalities (nephrotic stage, n = 14, remission stage, n = 10), 17 patients with FGS, and 20 healthy age-matched subjects were included in the present study. Urinary L-FABP levels were measured by enzyme-linked immunosorbent assay and compared. All patients with minor glomerular abnormalities at the nephrotic stage received prednisolone for 6 months, and all FGS patients received some form of immunosuppression therapy with prednisolone, cyclophosphamide or mizoribine for 12 months. LDL apheresis was performed in eight FGS patients with drug-resistant nephrotic syndrome. RESULTS: Urinary L-FABP levels were significantly higher in the 17 FGS patients (82.0 +/- 44.4 microg/g.Cr) than in the 24 patients with minor glomerular abnormalities (10.2 +/- 8.4 microg/g.Cr) (p < 0.01) and in the 20 healthy subjects (7.4 +/- 4.2 microg/g.Cr) (p < 0.01). Urinary L-FABP levels differed little between nephrotic stage and remission stage in patients with minor glomerular abnormalities. Urinary L-FABP levels were significantly higher in the eight drug-resistant FGS patients (122.6 +/- 78.4 microg/g.Cr) than in the nine drug-sensitive FGS patients (45.9 +/- 32.0 microg/g.Cr). Urinary L-FABP levels did not correlate with levels of other clinical markers including serum creatinine, urinary protein, and urinary N-acetyl-beta-D- glucosaminidase. In the eight drug-resistant FGS patients, LDL-apheresis significantly reduced urinary protein excretion (p < 0.01) and urinary L-FABP levels (p < 0.01). CONCLUSIONS: Urinary L-FABP may be a useful diagnostic indicator for differentiation between FGS and minor glomerular abnormalities. LDL apheresis may be effective in ameliorating tubulointerstitial lesions associated with FGS.

Preparation of anti-ras Mr 21,000 protein monoclonal antibodies and immunohistochemical analyses on expression of ras genes in human stomach and thyroid cancers.

Sixteen clones (RASK-1 to -16) of murine monoclonal antibodies were raised against ras Mr 21,000 protein (p21). The p21 produced by Escherichia coli with inserted v-Ki-ras genes was used as immunogen. RASK-1 was found to be specific for Ki-ras p21, whereas RASK-2 to -16 reacted with the p21s of Ki-, N-, and Ha-ras genes in both enzyme-linked immunosorbent and immunoblotting assays. Binding inhibition assays with biotinylated monoclonal antibodies by enzyme-linked immunosorbent assay showed that the monoclonal antibodies of the 16 clones included those binding to several mutually distinct sites on p21. The expressions of ras p21 in human stomach and thyroid tissues were examined with RASK-3, which reacted with all the Ki-, N-, and Ha-ras p21s immunohistochemically by the avidin-biotin peroxidase complex method. Formalin-fixed, paraffin-embedded tissues of 101 cases of stomach cancer, 53 cases of noncancerous stomach, 74 cases of cancer of the thyroid, and 59 cases of noncancerous thyroid were analyzed. In both the stomach and thyroid, cancer cells expressed p21 predominantly. Cells of cases with various noncancerous disorders as well as certain types of normal cells were also p21 positive. These findings suggest that precaution is required in use of p21 as a cancer marker. Expression of p21 was noted in moderately to well-differentiated stomach cancer, intestinal metaplasia, and atypical hyperplasia. This finding suggests that the appearance of p21 in stomach cancer may be initiated before cytological transformation.

An activated mutant of R-Ras inhibits cell death caused by cytokine deprivation in BaF3 cells in the presence of IGF-I.

R-Ras belongs to a family of low molecular weight GTP-binding proteins and exhibits 55% amino acid identity to H-Ras. It has been demonstrated that H-Ras inhibits cell death caused by interleukin-3 (IL-3) withdrawal in BaF3 cells (Kinoshita et al. (1995b); Terada et al. (1995)). In the present study, we examined whether R-Ras also rescues BaF3 cells from the factor-deprived cell death. To do this, several BaF3 transfectants were established, in which expression of wild-type as well as mutant R-Ras was regulated by an inducible promoter. Using these transfectants, we found that expression of an activated R-Ras mutant, R-Ras (Q87L), suppressed the death of IL-3-deprived BaF3 cells. On the other hand, expression of the wild-type and the dominant-negative mutant of R-Ras showed no inhibitory effect on cell death, indicating that R-Ras x GTP abrogated cell death caused by deprivation of IL-3. Furthermore, it was found that IGF-I in serum was required for the anti-apoptotic activity of R-Ras. Suppression of cell death by R-Ras(Q87L) was inhibited by wortmannin, LY294002 (phosphatidylinositol 3-kinase (PI3K) inhibitors), or PD98059 (inhibitor for MEK, a specific activator of mitogen-activated protein kinase (MAPK)). In addition, we have shown that, in HEK293 cells, R-Ras and IGF-I could activate MAPK synergistically. Also, PI3K activity was co-immunoprecipitated with an activated mutant of R-Ras. These results suggest that R-Ras in collaboration with IGF-I suppressed apoptotic cell death of BaF3 caused by IL-3 deprivation, presumably by modulating the activitites of MAPK and PI3K.

Positive regulation of skeletal myogenesis by R-Ras.

Most of the proteins in the Ras-family proteins, including Ras, Rap and TC21, have been reported to be strong inhibitors of skeletal myogenesis. Here we show that R-Ras, another member of this family, promotes terminal differentiation of C2C12 skeletal myoblasts. In contrast to Ras, which induced a markedly transformed phenotype of C2C12 cells, an activated mutant of R-Ras (R-RasQ87L) did not exhibit any inhibitory effect on the differentiation of C2C12 cells, but enhanced the formation of multinucleated myotubes. Although R-RasQ87L showed little effect on induction of two muscle-specific proteins, creatine kinase and myogenin, it prevented cell death during myoblast differentiation, probably through Akt activation and Bcl-xL induction. Motility of C2C12 cells, which may be involved in fusion of myoblasts, was also stimulated by R-RasQ87L. Furthermore, we observed a transient activation of endogenous R-Ras during differentiation of C2C12 cells. The ectopic expression of R-Ras GAP inhibited the differentiation. These results suggest that R-Ras has a positive effect on the terminal differentiation of myoblasts and may be involved in the program of skeletal myogenesis.

Isolation of a new protein factor required for activation of Raf-1 by Ha-Ras: partial purification from rat brain cytosols.

Ras-mediated signaling pathways play a critical role in cellular proliferation and differentiation. Although it has been demonstrated that Ras interacts with Raf-1 to stimulate the serine/threonine kinase activity of Raf-1, the precise mechanism by which Ras activates Raf-1 remains obscure. To address this question, we developed a cell-free system in which the activated form of H-Ras can induce Raf-1 activation. Using this system, we found the presence of a new protein factor, in cytosolic fractions of both human embryonic kidney 293 cells and rat brain tissues, that is required for Ras-dependent activation of Raf-1. The factor was purified from rat brain cytosols through successive column chromatographies on DEAE Sephacel, SP Sepharose and Sephacryl S-300. The approximate molecular weight of the activator was estimated as 400,000 by gel filtration. Its activity was sensitive to heat and trypsin treatments. The purified activator did not contain Src, 14-3-3, protein kinase C, JAK2 or Ksr-1, as judged by immunoblotting. Further characterization of the activator is underway.

A glutamic acid residue at position 31 of Ras protein is essential to the signal transduction for neurite outgrowth of PC12 cells and the stimulation of GTPase activity by GAPRas.

The roles of residues at positions 23-31 adjacent to the 'effector region' and residues at positions 61-65 in a phosphoryl binding loop of the human c-Ha-ras protein were studied by changing each residue of the normal (Gly-12 type) and oncogenic (Val-12 type) Ras proteins to the corresponding residue of the K-rev-1 protein. Firstly, the signal-transducing activities of the mutant Ras proteins of Val-12 type were examined by analysis of their ability to induce neurite outgrowth of phaeochromocytoma (PC12) cells upon expression of the mutant ras gene. Thus, replacement of Glu-31 by Lys was found to impair the signal-transducing activity of the oncogenic Ras protein. Furthermore, it was shown that expression of the Gly-12----Val/Glu-31----Lys mutant Ras protein in PC12 cells suppresses neurite outgrowth induced either by microinjection of the oncogenic Ras protein or by addition of nerve growth factor to the medium. As for the Glu-31----Lys mutant Ras protein (Gly-12 type), the GTPase activity in the presence of GTPase-activating protein for Ras (GAPRas) is much lower than that of the normal Ras protein, whereas the intrinsic GTPase activity is nearly the same as that of the normal Ras protein. Therefore, Gly-31 is one of the determinants for the signal transduction and the correct interaction with GAPRas. On the other hand, the GTPase activity of the Gln-61----Thr mutant Ras protein (Gly-12 type) is negligibly low both in the absence and in the presence of GAPRas.

Mutations that abolish the ability of Ha-Ras to associate with Raf-1.

Recent studies have revealed that Ras can associate physically with Raf. In the present study, we tested 34 mutants of Ha-Ras carrying substitution(s) in the region of residues 23-71 for their ability to associate with Raf-1. Mouse Ba/F3 cell lysates were incubated with each mutant Ras protein, in either the guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)- or the guanosine 5'-[beta-thio]diphosphate (GDP beta S)-bound form, and the anti-Ras antibody Y13-238. The immunoprecipitates were analysed for the presence of Raf-1 by Western blotting with an anti-Raf-1 antibody. Six mutants of Ras, E31K, P34G, T35S, D38N, D57A and A59T, failed to bind Raf-1. Mutations N26G, V29A, S39A, Y40W, R41A, V44A, V45E, L56A and T58A partially reduced the ability to bind Raf-1. All the other mutants could associate with Raf-1 with nearly the same efficiency as that of wild-type Ras. Thus, the Raf-I-binding ability of Ras appears to be affected by mutations in the N-terminal region, and in particular, by those in and neighboring the effector region (residues 32-40) and in the region (residues 56-59) flanking the N-terminal of Switch II. The abilities to bind Raf-1 and to induce neurite outgrowth of pheochromocytoma (PC) 12 cells correlate to each other for 22 Ras mutants. However, mutation A59T, which does not reduce the neurite-inducing or transforming activities, abolishes the ability to bind Raf-1. In contrast, mutations Y32F, K42A and L53A, which impair the neurite-inducing activity of Ras, have no effect on the Ras.Raf-1 association. Partially reduced Raf-1-binding ability was observed for mutants V29A, S39A, Y40W, R41A, V44A, L56A and T58A, which exhibit full neurite-inducing activity, and also for mutant V45E, which has no activity of neurite induction.

Activation of protein kinase C by intracellular free calcium in the motoneuron cell line NSC-19.

The relationship between intracellular free calcium ([Ca2+]i) and the activation of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) was investigated in the NSC-19 motoneuron cell line. Increased extracellular calcium ([Ca2+]o) up to 10 mM resulted in sustained elevations of [Ca2+]i. Control cell cultures (1.3 mM [Ca2+]o, [Ca2+]i = 83 +/- 17 nM) contained Ca2+- and PS/DO lipid-dependent PKC activity predominantly in the cytosol. However, elevation of [Ca2+]o up to 5 mM ([Ca2+]i = 232 +/- 24 nM) resulted in almost complete loss of cytosolic PKC activity. Cells incubated in 10 mM [Ca2+]o ([Ca2+]i = 365 +/- 13 nM) showed increased levels of both cytosolic and membrane PKC activity compared to control. These alterations in PKC activity appeared to be translocation-independent, since PKC protein levels were unchanged as demonstrated by Western blotting analysis. When cells were exposed to 25 or 50 mM [Ca2+]o, [Ca2+]i rose transiently to over 600 and 900 nM, respectively, and then returned to near basal values. Under these conditions, total PKC activity decreased, and increased amounts of the catalytic fragment of PKC, protein kinase M, were generated. Extracts from cells exposed to [Ca2+]o between 1.3 and 25 mM did not differ significantly in the levels of measurable CaMKII activity 10 min following the change in [Ca2+]o.

A site-directed mutagenesis study on the role of isoleucine-23 of human epidermal growth factor in the receptor binding.

The isoleucine-23 residue of human epidermal growth factor (hEGF) was substituted by a variety of amino acid residues and the receptor-binding activities of variant hEGFs were determined by the use of human KB cell. Tight receptor binding was found of variants with hydrophobic amino acid residues in position 23. The size of the isoleucine residue was nearly optimum for the receptor binding as compared with other hydrophobic residues. The structure analysis by two-dimensional nuclear magnetic resonance spectroscopy showed that the substitution at position 23 only slightly affected the tertiary structure of hEGF. These indicate that the side chain of isoleucine residue in position 23, which is exposed on the protein surface, directly binds to a hydrophobic pocket of the receptor.

ECM gene expression and its modulation by insulin in diabetic rats.

The steady-state levels of mRNA encoding for the alpha 1(IV) collagen chain, laminin B1 and B2 chains, basement membrane HSPG, and alpha 1(I) and alpha 1(III) collagen chains were examined in rat glomeruli at 4, 12, and 24 wk after injection of STZ. The mRNA levels for the alpha 1(IV) collagen chain, laminin B1 and B2 chains, and alpha 1(I) and alpha 1(III) collagen chains increased significantly with age in the STZ-induced diabetic rats before morphological thickening of basement membrane occurred. In contrast, the mRNA levels for HSPG decreased markedly 4 wk after STZ injection and then increased with age compared with those for control rats. The mRNA levels for these ECM components showed a continuous decline with age in controls. Treating the diabetic rats with insulin for 4 wk ameliorated the abnormally regulated ECM gene expression in the glomeruli. These data suggest that the abnormal regulation of ECM gene expression in the glomeruli may contribute to the expansion of mesangial matrix and basement membrane thickening in diabetic rats, and that hyperglycemia may play a role in the abnormal ECM gene expression.

Effect of a specific endothelin receptor A antagonist on mRNA levels for extracellular matrix components and growth factors in diabetic glomeruli.

We have previously reported that the mRNA levels of extracellular matrix (ECM) components including alpha 1(I), alpha 1(III), and alpha 1(IV) collagen chains, laminin B1 and B2 chains, and growth factors including tumor necrosis factor (TNF)-alpha, platelet-derived growth factor (PDGF)-B chain, transforming growth factor (TGF)-beta, and basic fibroblast growth factor (FGF) all increase with age in diabetic glomeruli. The present study was designed to assess whether glomerular expression of these mRNAs in rat diabetic glomeruli is affected by a specific endothelin receptor A antagonist, FR139317. Diabetes was produced by streptozotocin injection, and animals were divided into four groups: untreated diabetic rats, FR139317-treated diabetic rats, control nondiabetic rats, and FR139317-treated control rats. FR139317 treatment was continued for 24 weeks. FR139317 attenuated the rise in creatinine clearance (P < 0.01) and reduced urinary protein excretion (P < 0.01) in diabetic rats, but did not affect blood pressure. FR139317 attenuated the increases in glomerular mRNA levels of alpha 1(I) (P < 0.01), alpha 1(III) (P < 0.01), and alpha 1(IV) (P < 0.01) collagen chains, laminin B1 (P < 0.01) and B2 (P < 0.01) chains, TNF-alpha (P < 0.01), PDGF-B (P < 0.01), TGF-beta (P < 0.001) and basic FGF (P < 0.01) in diabetic rats. However, FR139317 did not affect these mRNA levels in glomeruli of control rats. These findings suggest that FR139317 may be useful in the treatment of diabetic glomerulopathy by reducing mRNA levels of ECM components and growth factors.

mRNA expression of growth factors in glomeruli from diabetic rats.

Evaluations of glomerular mRNA levels encoding for PCNA, TNF-alpha, PDGF-A and -B chains, TGF-beta, IGF-I, bFGF, and EGF were made at 4, 12, and 24 wk after injection of STZ in Sprague-Dawley rats. The mRNA levels for PCNA, TNF-alpha, PDGF-B chain, TGF-beta, and bFGF increased with age in STZ-induced diabetic rats. At 24 wk after STZ injection, mRNA levels for PCNA, TNF-alpha, PDGF-B chain, TGF-beta, and bFGF were increased 3.8-fold, (P < 0.01), 4.2-fold (P < 0.01), 4.0-fold (P < 0.01), 5.2-fold (P < 0.001), and 3.6-fold (P < 0.01), respectively, in the glomeruli of diabetic rats when compared with control rats. In contrast, mRNA levels for IGF-I, PDGF-A chain, and EGF were not altered in glomeruli from diabetic and control rats throughout the experimental period. Insulin treatment partially ameliorated the increase in mRNA levels for PCNA, TNF-alpha, PDGF-B chain, TGF-beta, and bFGF in the glomeruli of diabetic rats. These data indicate that alterations in growth factor mRNA levels in glomeruli may be a manifestation of diabetic nephropathy, and that hyperglycemia or insulin deficiency may play a role in abnormal growth factor gene regulation.