The bone morphogenetic proteins (BMPs) profoundly affect embryonic development, differentiation and disease. BMP signaling is suppressed by cysteine-rich domain proteins, such as chordin, that sequester ligands from the BMP receptor. We describe a novel protein, KCP, with 18 cysteine-rich domains. Unlike chordin, KCP enhances BMP signaling in a paracrine manner. Smad1-dependent transcription and phosphorylated Smad1 (P-Smad1) levels are increased, as KCP binds to BMP7 and enhances binding to the type I receptor. In vivo, Kcp(-/-) mice are viable and fertile. Because BMPs have a pivotal role in renal disease, we examined the phenotype of Kcp(-/-) mice in two different models of renal injury. Kcp(-/-) animals show reduced levels of P-Smad1, are more susceptible to developing renal interstitial fibrosis, are more sensitive to tubular injury and show substantial pathology after recovery. The data indicate an important role for KCP in attenuating the pathology of renal fibrotic disease.

Reducing luminal NaCl concentration in the macula densa region of the nephron stimulates renin secretion, and this response is blocked by a specific inhibitor of cyclooxygenase-2 (COX-2) (Traynor, T. R., Smart, A., Briggs, J. P., and Schnermann, J. (1999) Am. J. Physiol. Renal Physiol. 277, F706 -710). To study whether low NaCl activates COX-2 activity or expression we clonally derived a macula densa cell line (MMDD1 cells) from SV-40 transgenic mice using fluorescence-activated cell sorting of renal tubular cells labeled with segment-specific fluorescent lectins. MMDD1 cells express COX-2, bNOS, NKCC2, and ROMK, but not Tamm-Horsfall protein, and showed rapid 86 Rb ؉ uptake that was inhibited by a reduction in NaCl concentration and by bumetanide or furosemide. Isosmotic exposure of MMDD1 cells to low NaCl (60 mM) caused a prompt and time-dependent stimulation of prostaglandin E 2 (PGE 2 ) release that was prevented by the COX-2 specific inhibitor NS-398 (10 M). Reducing NaCl to 60 and 6 mM for 16 h increased COX-2 expression in a chloride-dependent fashion. Low NaCl phosphorylated p38 kinase within 30 min and ERK1/2 kinases within 15 min without changing total MAP kinase levels. Low NaCl-stimulated PGE 2 release and COX-2 expression was inhibited by SB 203580 and PD 98059 (10 M), inhibitors of p38 and ERK kinase pathways. We conclude that low chloride stimulates PGE 2 release and COX-2 expression in MMDD1 cells through activation of MAP kinases.

Cyclic mechanical stretch of bladder smooth muscle cells (SMC) increases rates of DNA synthesis and stimulates transcription of the gene for heparin-binding epidermal growth factor-like growth factor (HB-EGF), an ErbB1/EGF receptor ligand that has been linked to hypertrophic bladder growth. In this study we sought to clarify the signaling pathways responsible for mechanotransduction of the stretch stimulus. HB-EGF mRNA levels, DNA synthesis, and AP-1/Ets DNA binding activities were induced by repetitive stretch of primary culture rat bladder SMC. Inhibitors of the p38 SAPK2 pathway, the angiotensin receptor type 1 (AT1), and the ErbB2 tyrosine kinase reduced each of these activities, while an inhibitor of the extracellular signal-regulated kinase mitogen-activated protein kinase (Erk-MAPK) pathway had no effect. Stretch rapidly activated stress-activated protein kinase 2 (p38 SAPK2) and Jun NH(2)-terminal kinase (JNK)/SAPK pathways but not the Erk-MAPK pathway and induced ErbB2 but not ErbB1 phosphorylation. Angiotensin II (ANG II) a bladder SMC mitogen previously linked to the stretch response, did not activate ErbB2, and ErbB2 activation occurred in response to stretch in the presence of an ANG receptor inhibitor, indicating that activation of the AT1-mediated pathway and the ErbB2-dependent pathway occurs by independent mechanisms. p38 SAPK2 and JNK/SAPK signaling also appeared to be independent of the ErbB2 and AT1 pathways. These findings indicate that stretch-stimulated DNA synthesis and gene expression in normal bladder SMC occur via multiple independent receptor systems (e.g., AT1 and ErbB2) and at least one MAPK pathway (p38 SAPK2). Further, we show that the Erk-MAPK pathway, which in most systems is linked to receptor-dependent cell growth responses, is not involved in progression to DNA synthesis or in the response of the HB-EGF gene to mechanical forces.

Mechanical induction of growth factor synthesis may mediate adaptive responses of smooth muscle cells (SMC) to increases in physical load. We previously demonstrated that cyclic mechanical stretch induces expression of the SMC, fibroblast, and epithelial cell mitogen heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bladder SMC, an observation that suggests that this growth factor may be involved in compensatory bladder hypertrophy. In the present study we provide evidence that the activator protein-1 (AP-1) transcription factor plays a critical role in this mechanoinduction process. Rat bladder SMC were transiently transfected with a series of 5′ deletion mutants of a promoter-reporter construct containing 1.7 kb of the mouse HB-EGF promoter that was previously shown to be stretch responsive. The stretch-mediated increase in promoter activity was completely ablated with deletion of nucleotide positions −1301 to −881. Binding of AP-1, as evaluated by electrophoretic mobility shift assay, to a synthetic oligonucleotide containing an AP-1 binding site increased in response to stretch, and binding was inhibited by excess unlabeled DNA corresponding to nucleotides −993 to −973 from the HB-EGF promoter, a region that contains a previously recognized composite AP-1/Ets site. Stretch-induced promoter activity was significantly inhibited by site-directed mutagenesis of the AP-1 or Ets components of this site. Consistent with the promoter and gel-shift studies, curcumin, an inhibitor of AP-1 activation, suppressed the HB-EGF mRNA induction after stretch. Stretch also specifically increased mRNA levels for matrix metalloproteinase (MMP)-1, the promoter of which contains a functional AP-1 element, but not for MMP-2, the promoter of which does not contain an AP-1 element. The stretch response of the MMP-1 gene was also completely inhibited by curcumin. Collectively, these findings indicate that AP-1-mediated transcription plays an important role in the regulation of gene expression in bladder muscle in response to mechanical forces.

Studies were performed to investigate the regulatory mechanism of bladder cyclooxygenase-2 (COX-2) expression after outlet obstruction. In situ hybridization of murine bladder tissues using COX-2-specific riboprobes demonstrated that COX-2 expression was induced predominantly in the bladder smooth muscle cells after outlet obstruction. To study the effect of increased mechanical stretch on COX isoform expression, cultured rat bladder smooth muscle cells were grown on silicone elastomer-bottomed plates coated with collagen type I and were subjected to continuous cycles of stretch/relaxation for variable duration. COX-1 mRNA levels did not change with stretch. COX-2 expression increased in a time-dependent manner after stretch, with maximal mRNA and protein levels occurring after 4 h. PGE2 levels increased more than 40-fold in the culture media after stretch, consistent with increased COX activity, and this was reduced to near completion in the presence of a COX-2 inhibitor, NS-398. Exposure to stretch over a 48-h period induced a 4.7 ± 0.6-fold increase in tritiated thymidine incorporation rate. This increase in DNA synthesis was markedly suppressed when the cells were stretched in the presence of NS-398. We conclude that in bladder obstruction COX-2 activation occurs predominantly in the smooth muscle cells in response to mechanical stretch. Our findings also suggest that stretch-activated COX-2 expression may participate in bladder smooth muscle cell proliferation and thereby play a role in pathological bladder wall thickening after obstruction.

Cultured rat bladder smooth muscle cells (SMC) were grown on collagen-coated silicone membranes and subjected to continuous cycles of stretch-relaxation. Semiquantitative RT-PCR analysis revealed a time-dependent increase in heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) mRNA levels after stretch, with maximal levels appearing after 4 h. Immunostaining for proHB-EGF revealed higher levels of HB-EGF protein in the stretched than in the nonstretched SMC. The ANG II receptor type 1 antagonist losartan markedly suppressed stretch-activated HB-EGF expression. ANG II levels were 3.3-fold higher in the stretch- than in the non-stretch-conditioned media. Stretch stimulation of bladder SMC that had been transiently transfected with an HB-EGF promoter-luciferase expression construct resulted in an 11-fold increase in reporter activity. Mechanical stretch induced a 4.7-fold increase in tritiated thymidine incorporation rate, and this was reduced by 25% in the presence of losartan. We conclude that mechanical stretch activates HB-EGF gene expression in bladder SMC and that this is mediated in part by autocrine ANG II secretion.

Studies were undertaken to assess expression of inducible cyclooxygenase (COX)-2 in bladder during fetal development and COX-1 and COX-2 expression after outlet obstruction. Bladder tissue or bladder progenitor tissue was harvested from CD-1 murine embryos at embryonic days 11.5( E11.5), E14.5, E17.5, E20.5 (newborn), and from adult. Bladder obstruction was created in adult female mice by ligating the urethra, and bladders were harvested after 3–24 h of obstruction. Gene expression was assessed by semiquantitative reverse transcription-polymerase chain reaction and Western blotting. COX-2 was highly expressed at the early stages of bladder development and declined progressively throughout gestation. In adult bladder, both COX-1 and COX-2 were detectable at low levels under basal conditions. An ∼30-fold increase in COX-2 mRNA was seen after 24 h of obstruction. In contrast, COX-1 did not change with obstruction. COX-2 mRNA levels peaked at 6 h of obstruction. In regional bladder-distention models, COX-2 induction was confined to the area of distention. Bladder outlet obstruction stimulates COX-2 expression dramatically, reactivating a gene that is highly expressed during fetal development.

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