Lysyl oxidase regulates MMTV promoter: indirect evidence of histone H1 involvement.

Lysyl oxidase (LOX) is the enzyme that facilitates the cross-linking of collagen and elastin, although other functions for this enzyme have been indicated. Of these other functions, we describe herein the ability of LOX to regulate several gene promoters, like collagen III, elastin, and cyclin D1. We have previously demonstrated a specific binding between LOX and histone H1, in vitro. Therefore, we investigated whether LOX would affect the mouse mammary tumor virus (MMTV) promoter and its glucocorticoid regulation, which depends on the phophorylation status of histone H1. Our results show that the over-expression of recombinant human LOX was able to trigger MMTV activity, both in the presence and absence of glucocorticoids. Moreover, we demonstrated that histone H1 from cells expressing recombinant LOX contained isodesmosine and desmosine, indicating specific lysyl-oxidase-dependent lysine modifications. Finally, we were able to co-immunoprecipitate the exogenous LOX and histone H1 from the LOX transfected cells. The data are compatible with a decreased positive charge of histone H1, owing to deamination by LOX of its lysine residues. This event would favor H1 detachment from the target DNA, and consequent opening of the MMTV promoter structure to the activating transcription factors. The presented data, therefore, suggest a possible histone-H1-dependent mechanism for the modulation of MMTV promoter by LOX.

Regulation of elastin promoter by lysyl oxidase and growth factors: cross control of lysyl oxidase on TGF-beta1 effects.

Lysyl oxidase (LOX) plays a key role in the maturation of the extra-cellular matrix, by inducing the formation of lysyl cross-links in collagen and elastin molecules. Beside its enzymic activity, LOX is able to regulate the promoter of collagen III, one of its natural substrates. In this paper we demonstrated that LOX regulates also the promoter of elastin, inducing an important activation of its activity. In order to define the pathways used by LOX to achieve its effect, we activated some of the main fibrogenic signal pathways and studied the consequences on LOX effects on the promoter. TGF-beta1 activated most of the elastin promoter constructs that we studied, except for an inhibitory region contained in the region between -1500 and -1000 bp. The treatment with TGF-beta1 abolished completely the activation induced by LOX. LOX-over-expression coupled with TGF treatment abolished both effects in the -500 bp region. The treatment with CTGF also inhibited LOX effect, although to a lesser extent. However, CTGF behaved quite differently from TGF-beta1 suggesting that it is not necessarily the mediator of TGF effects. Basic FGF, the other fibrogenic factor that we tested, again abolished LOX-dependent activation, but by itself did not affect elastin promoter activity. Because TGF-beta1 activating effects, we used EMSA to examine the transcription factor binding patterns in presence of LOX, TGF-beta1 or both. The study showed that LOX reverted the patterns of several DNA-protein complexes along the 1.5 kb of the studied promoter region. Most were affected by both LOX and TGF-beta1, while on some only TGF-beta1 was effective. LOX presence mostly inhibited the TGF-regulated complexes. Many of those included SMAD transcription factors. Two more restricted regions binding AP1 and SMAD were identified as mediators of LOX effects and of LOX and TGF-beta1 cross-inhibition.

Rare functional variants of podocin (NPHS2) promoter in patients with nephrotic syndrome.

Podocin (NPHS2) is a component of the glomerular slit-diaphragm, with major regulatory functions in renal permeability of proteins. Loss of podocin and decrease in resynthesis may influence the outcome of proteinuric renal disease such as segmental glomerulosclerosis (FSGS), and promoter functionality plays a key role in this process. NPHS2 promoter variants with functional activity may be a part of the problem of podocin resynthesis. We sequenced NPHS2 promoter region from -628 to ATG in a large cohort of 260 nephrotic patients (161 with FSGS) who were presenting proteinuria from moderate to severe and were receiving or had received modular therapies according to their sensitivity to steroids and other immune modulators. Three sequence variants (-236C>T, -52C>G, -26C>G) were identified in our study population that gave an allele frequency below 1% (5 patients out of 520 alleles). Functional implications were shown for each variants that were most evident for -52C>G and -26C>G (-50% of luciferase expression compared to the wild-type sequence, p < 0.01). Consensus analysis for homology of the -52 region with regulatory factors revealed homology for USF1 and the sum of experiments with gel retardation and with cells silenced for USF1 confirmed that this factor regulates NPHS2 expression at this site. In conclusion, three functional variants in NPHS2 promoter have been identified in a large cohort of patients with nephrotic syndrome and FSGS that have a frequency <1%. One of these (i.e., -52C>G) is associated with a poor clinical outcome and evolution to end-stage renal failure. USF1 was identified as the transcriptional factor regulating NPHS2 at this site. Even if not sufficient to cause FSGS per se, these variants could represent modifiers for severity and/or progression of the disease.

beta-catenin signaling and regulation of cyclin D1 promoter in NRK-49F cells transformed by down-regulation of the tumor suppressor lysyl oxidase.

Lysyl oxidase is the enzyme that is essential for collagen and elastin cross-linking. Previous investigations showed that lysyl oxidase is down-regulated in many human tumors and ras-transformed cells. Recently, we proved that antisense down-regulation of lysyl oxidase in NRK-49F cells induced phenotypic changes and oncogenic transformation, characterized by p21(ras) activation and beta-catenin/cyclin D1 up-regulation. In the present paper, we examined beta-catenin intracellular distribution and its association with E-cadherin. We observed an increased association between E-cadherin and beta-catenin in the lysyl-oxidase down-regulated cells during serum starvation. Moreover, we found that beta-catenin cytoplasmic and nuclear levels were increased, suggesting a failure of its down-regulation by the APC-GSK-3beta system, in particular the GSK-3beta phosphorylation of ser-33/37 and thr-41 of beta-catenin. Finally, we investigated the mechanisms leading to the observed cyclin D1 up-regulation. We showed that in the antisense lysyl oxidase cells the cyclin D1 promoter was activated through the LEF and the ATF/CRE sites in the proximal promoter. While the promoter activation through LEF is compatible with beta-catenin signaling, we investigated the possibility that the CRE-dependent activation might be linked to the down-regulation of lysyl oxidase. In fact, up-regulation of lysyl oxidase in a COS-7 cell model showed a significant diminution of the CREB protein binding to the cyclin D1 promoter, leading to a dramatic inhibition of its activity and a significant down-regulation of cyclin D1 protein level in vivo. Finally, our study describes some major anomalies occurring in lysyl oxidase down-regulated fibroblasts, related to beta-catenin signaling and cyclin D1 expression.

Altered adhesion features and signal transduction in NRK-49F cells transformed by down-regulation of lysyl oxidase.

Lysyl oxidase (LOX) down-regulation induced an oncogenic phenotype in NRK-49F. This event was accompanied by a constitutive activation of ras oncogene and down-regulation of PDGF beta receptor, among other important phenotypic and molecular modifications. In the present paper we show that ras activation is not accompanied by a constitutive activation of the MAP kinases as expected. Surprisingly, even if MAPK-independent, ras activation was accompanied by a constitutive Ser(63) and Ser(73) phosphorylation of c-jun, a further downstream target of ras. Although rare, this ras alternative pathway has been described. Since ras alone is seldom able to trigger cell transformation and the transformed phenotype showed clearly an abnormal adhesion pattern, we investigated the main molecules involved in cell-cell adhesion. In fact, we found that beta-catenin was up-regulated, escaping the glycogen synthase kinase-3 beta (GSK-3 beta) control, through unclear mechanisms. Its nuclear accumulation was accompanied by an up-regulation of cyclin D1, as classically described in the activation of the Wnt/beta-catenin signal pathway. We believe that the resulting up-regulation of cyclin D1 acted in synergy with ras to induce the cell transformation.

Demonstration of in vitro interaction between tumor suppressor lysyl oxidase and histones H1 and H2: definition of the regions involved.

Lysyl oxidase (LOX) is the enzyme that cross-links extracellular collagen and tropoelastin and is involved in tumor suppressor activity. Based on the existent homologies between lysine-rich regions of tropoelastin and the "lysine-rich" histone H1, we tested the possibility that H1 could be a new nuclear target. Our study shows that LOX could actually interact specifically not only with histone H1, but also with histone H2. Mechanisms and significance of these interactions are discussed in detail.