Mattias C. U. Gustafsson

Activation of peroxisome proliferator-activated receptor delta stimulates the proliferation of human breast and prostate cancer cell lines

The nuclear receptor peroxisome proliferator-activated receptor delta [PPARdelta/beta (NR1C2)] has been implicated in colorectal carcinogenesis by various molecular genetic observations. These observations have recently been supported by studies of activation of PPARdelta by pharmacological agents. Here we present the first report of the stimulation of breast and prostate cancer cell growth using PPARdelta selective agonists. Activation of PPARdelta with compound F stimulated proliferation in breast (T47D, MCF7) and prostate (LNCaP, PNT1A) cell lines, which are responsive to sex hormones. Conversely, we have found that several steroid-independent cell lines, including colon lines, were unresponsive to compound F. These findings were confirmed with an additional high-affinity PPARdelta agonist, GW501516. Conditional expression of PPARdelta in MCF7 Tet-On cells resulted in a doxycycline-enhanced response to GW501516, thus providing direct genetic evidence for the role of PPARdelta in the proliferative response to this drug. Activation of PPARdelta in T47D cells resulted in increased expression of the proliferation marker Cdk2 and also vascular endothelial growth factor alpha (VEGFalpha) and its receptor, FLT-1, thus, suggesting that PPARdelta may initiate an autocrine loop for cellular proliferation and possibly angiogenesis. Consistent with this hypothesis, we demonstrated a pro-proliferative effect of GW501516 on human umbilical vein endothelial cell cultures and found that GW501516 also regulated the expression of VEGFalpha and FLT-1 in these cells. Our observations provide the first evidence that activation of PPARdelta can result in increased growth in breast and prostate cancer cell lines and primary endothelial cells and supports the possibility that PPARdelta antagonists may be of therapeutic value in the treatment of breast and prostate cancer.

A genetic basis of susceptibility to acute pyelonephritis.

Background For unknown reasons, urinary tract infections (UTIs) are clustered in certain individuals. Here we propose a novel, genetically determined cause of susceptibility to acute pyelonephritis, which is the most severe form of UTI. The IL-8 receptor, CXCR1, was identified as a candidate gene when mIL-8Rh mutant mice developed acute pyelonephritis (APN) with severe tissue damage. Methods and Findings We have obtained CXCR1 sequences from two, highly selected APN prone patient groups, and detected three unique mutations and two known polymorphisms with a genotype frequency of 23% and 25% compared to 7% in controls (p<0.001 and p<0.0001, respectively). When reflux was excluded, 54% of the patients had CXCR1 sequence variants. The UTI prone children expressed less CXCR1 protein than the pediatric controls (p<0.0001) and two sequence variants were shown to impair transcription. Conclusions The results identify a genetic innate immune deficiency, with a strong link to APN and renal scarring.

Reduced Toll-Like Receptor 4 Expression in Children with Asymptomatic Bacteriuria

Toll-like receptor (TLR) 4 is essential for the defense against infection with gram-negative pathogens, but reduced TLR4 expression has not been linked to altered disease susceptibility in humans. In mice, Tlr4 controls the mucosal response to Escherichia coli urinary tract infections. Inactivation of mouse Tlr4 causes an asymptomatic carrier state resembling asymptomatic bacteriuria (ABU). The present study compared neutrophil TLR4 expression levels between children with ABU (n=17) and age-matched control subjects (n=24), and significantly lower levels were detected in the patients with ABU. We also found elevated levels of the TLR4 adaptor protein TRIF and reduced levels of the TLR4-inhibitor SIGIRR in the patients with ABU, but MyD88 and TRAM levels were not significantly altered. Altered TLR4 and adaptor protein expression might impair TLR4 signaling and explain the weak mucosal response to urinary tract infection in patients who develop ABU rather than symptomatic disease.

Toll-like receptor 4 promoter polymorphisms: common TLR4 variants may protect against severe urinary tract infection.

Background Polymorphisms affecting Toll-like receptor (TLR) structure appear to be rare, as would be expected due to their essential coordinator role in innate immunity. Here, we assess variation in TLR4 expression, rather than structure, as a mechanism to diversify innate immune responses. Methodology/Principal Findings We sequenced the TLR4 promoter (4,3 kb) in Swedish blood donors. Since TLR4 plays a vital role in susceptibility to urinary tract infection (UTI), promoter sequences were obtained from children with mild or severe disease. We performed a case-control study of pediatric patients with asymptomatic bacteriuria (ABU) or those prone to recurrent acute pyelonephritis (APN). Promoter activity of the single SNPs or multiple allelic changes corresponding to the genotype patterns (GPs) was tested. We then conducted a replication study in an independent cohort of adult patients with a history of childhood APN. Last, in vivo effects of the different GPs were examined after therapeutic intravesical inoculation of 19 patients with Escherichia coli 83972. We identified in total eight TLR4 promoter sequence variants in the Swedish control population, forming 19 haplotypes and 29 genotype patterns, some with effects on promoter activity. Compared to symptomatic patients and healthy controls, ABU patients had fewer genotype patterns, and their promoter sequence variants reduced TLR4 expression in response to infection. The ABU associated GPs also reduced innate immune responses in patients who were subjected to therapeutic urinary E. coli tract inoculation. Conclusions The results suggest that genetic variation in the TLR4 promoter may be an essential, largely overlooked mechanism to influence TLR4 expression and UTI susceptibility.

TLR- and CXCR1-dependent innate immunity: insights into the genetics of urinary tract infections.

The susceptibility to urinary tract infection (UTI) is controlled by the innate immune response and Toll like receptors (TLRs) are the sentinels of this response. If productive, TLR4 signalling may initiate the symptomatic disease process. In the absence of TLR4 signalling the infected host instead develops an asymptomatic carrier state. The activation of mucosal TLR4 is also influenced by the properties of the infecting strain, and pathogens use their virulence factors to trigger ‘pathogen‐specific’ TLR4 responses in the urinary tract but do not respond to the asymptomatic carrier strains in patients with asymptomatic bacteriuria (ABU). The TLR4 dependence has been demonstrated in mice and the relevance of low TLR4 function for protection for human disease was recently confirmed in children with asymptomatic bacteriuria, who expressed less TLR4 than age matched controls. Functional chemokines and functional chemokine receptors are crucial for neutrophil recruitment, and for the neutrophil dependent bacterial clearance. Interleukin (IL)‐8 receptor deficient mice develop acute septic infections and chronic tissue damage, due to aberrant neutrophil function. This mechanism is relevant for human UTI as pyelonephritis prone children express low levels of the human CXCL8 (Il‐8) receptor, CXC chemokine receptor 1 (CXCR1) and often have heterozygous CXCR1 polymorphisms.

Activation of PPARβ/δ Causes a Psoriasis-Like Skin Disease In Vivo

Background Psoriasis is one of the most frequent skin diseases world-wide. The disease impacts enormously on affected patients and poses a huge financial burden on health care providers. Several lines of evidence suggest that the nuclear hormone receptor peroxisome proliferator activator (PPAR) β/δ, known to regulate epithelial differentiation and wound healing, contributes to psoriasis pathogenesis. It is unclear, however, whether activation of PPARβ/δ is sufficient to trigger psoriasis-like changes in vivo. Methodology/Principal Findings Using immunohistochemistry, we define the distribution of PPARβ/δ in the skin lesions of psoriasis. By expression profiling, we confirm that PPARβ/δ is overexpressed in the vast majority of psoriasis patients. We further establish a transgenic model allowing inducible activation of PPARβ/δ in murine epidermis mimicking its distribution in psoriasis lesions. Upon activation of PPARβ/δ, transgenic mice sustain an inflammatory skin disease strikingly similar to psoriasis, featuring hyperproliferation of keratinocytes, dendritic cell accumulation, and endothelial activation. Development of this phenotype requires the activation of the Th17 subset of T cells, shown previously to be central to psoriasis. Moreover, gene dysregulation in the transgenic mice is highly similar to that in psoriasis. Key transcriptional programs activated in psoriasis, including IL1-related signalling and cholesterol biosynthesis, are replicated in the mouse model, suggesting that PPARβ/δ regulates these transcriptional changes in psoriasis. Finally, we identify phosphorylation of STAT3 as a novel pathway activated by PPARβ/δ and show that inhibition of STAT3 phosphorylation blocks disease development. Conclusions Activation of PPARβ/δ in the epidermis is sufficient to trigger inflammatory changes, immune activation, and signalling, and gene dysregulation characteristic of psoriasis.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) triggers autophagic tumor cell death.

HAMLET, a complex of partially unfolded α‐lactalbumin and oleic acid, kills a wide range of tumor cells. Here we propose that HAMLET causes macroautophagy in tumor cells and that this contributes to their death. Cell death was accompanied by mitochondrial damage and a reduction in the level of active mTOR and HAMLET triggered extensive cytoplasmic vacuolization and the formation of double‐membrane‐enclosed vesicles typical of macroautophagy. In addition, HAMLET caused a change from uniform (LC3‐I) to granular (LC3‐II) staining in LC3‐GFP‐transfected cells reflecting LC3 translocation during macroautophagy, and this was blocked by the macroautophagy inhibitor 3‐methyladenine. HAMLET also caused accumulation of LC3‐II detected by Western blot when lysosomal degradation was inhibited suggesting that HAMLET caused an increase in autophagic flux. To determine if macroautophagy contributed to cell death, we used RNA interference against Beclin‐1 and Atg5. Suppression of Beclin‐1 and Atg5 improved the survival of HAMLET‐treated tumor cells and inhibited the increase in granular LC3‐GFP staining. The results show that HAMLET triggers macroautophagy in tumor cells and suggest that macroautophagy contributes to HAMLET‐induced tumor cell death.

Fatty-acid-displaced transcriptional repressor, a conserved regulator of cytochrome P450 102 transcription in Bacillus species.

Abstract. Bacillus subtilis strain 168 encodes two flavocytochromes P450, Cyp102A2 and Cyp102A3. The cyp102A3 gene is preceded by, and organized in an operon with, a gene for a transcriptional regulator, encoded by fatR. The paralogous gene, cyp102A2, is most likely transcribed as a mono-cistronic message. We show that fatR encodes a protein that binds to an operator sequence that is present upstream of its own reading frame, thereby repressing the expression of the fatR-cyp102A3 operon. Unsaturated fatty acids and phytanic acid have the capacity to interact with FatR and to abrogate its binding to the operator sequence.

Histone Deacetylase Inhibitors Promote the Tumoricidal Effect of HAMLET

Histone deacetylase inhibitors (HDIs) and HAMLET (human alpha-lactalbumin made lethal to tumor cells) interact with histones, modify the structure of chromatin, and trigger tumor cell death. This study investigated how the combination of HDIs and HAMLET influences cell viability, histone acetylation, and DNA integrity. The pretreatment of tumor cells with HDIs was shown to enhance the lethal effect of HAMLET and the histone hyperacetylation response to HDIs increased even further after HAMLET treatment. HDIs and HAMLET were shown to target different histone domains as HAMLET bound tailless core histones, whereas HDIs modify the acetylation of the histone tail. DNA damage in response to HAMLET was increased by HDIs. The DNA repair response (p21WAFI expression) was induced by both agonists but abolished when the two agonists were combined. The results suggest that the synergy of HDIs and HAMLET is based on different but converging death pathways, both involving chromatin alterations. We speculate that HAMLET and HDIs might be combined to promote tumor cell death in vivo.

Ligand Modulated Antagonism of PPAR gamma by Genomic and Non-Genomic Actions of PPAR delta

Background Members of the Peroxisome Proliferator Activated Receptor, PPAR, subfamily of nuclear receptors display complex opposing and overlapping functions and a wide range of pharmacological and molecular genetic tools have been used to dissect their specific functions. Non-agonist bound PPARδ has been shown to repress PPAR Response Element, PPRE, signalling and several lines of evidence point to the importance of PPARδ repressive actions in both cardiovascular and cancer biology. Methodology/Principal Findings In this report we have employed transient transfections and luciferase reporter gene technology to study the repressing effects of PPARδ and two derivatives thereof. We demonstrate for the first time that the classical dominant negative deletion of the Activation Function 2, AF2, domain of PPARδ show enhanced repression of PPRE signalling in the presence of a PPARδ agonist. We propose that the mechanism for the phenomenon is increased RXR heterodimerisation and DNA binding upon ligand binding concomitant with transcriptional co-repressor binding. We also demonstrated ligand-dependent dominant negative action of a DNA non-binding derivative of PPARδ on PPARγ1 signalling. This activity was abolished upon over-expression of RXRα suggesting a role for PPAR/cofactor competition in the absence of DNA binding. Conclusions/Significance These findings are important in understanding the wide spectrum of molecular interactions in which PPARδ and PPARγ have opposing biological roles and suggest novel paradigms for the design of different functional classes of nuclear receptor antagonist drugs.