Initiation of human colon cancer cell proliferation by trypsin acting at protease-activated receptor-2

The protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin. We investigated the expression of PAR-2 and the role of trypsin in cell proliferation in human colon cancer cell lines. A total of 10 cell lines were tested for expression of PAR-2 mRNA by Northern blot and RT-PCR. PAR-2 protein was detected by immunofluorescence. Trypsin and the peptide agonist SLIGKV (AP2) were tested for their ability to induce calcium mobilization and to promote cell proliferation on serum-deprived cells. PAR-2 mRNA was detected by Northern blot analysis in 6 out of 10 cell lines [HT-29, Cl.19A, Caco-2, SW480, HCT-8 and T84]. Other cell lines expressed low levels of transcripts, which were detected only by RT-PCR. Further results were obtained with HT-29 cells: (1) PAR-2 protein is expressed at the cell surface; (2) an increase in intracellular calcium concentration was observed upon trypsin (1–100 nM) or AP2 (10–100 μM) challenges; (3) cells grown in serum-deprived media supplemented with trypsin (0.1–1 nM) or AP2 (1–300 μM) exhibited important mitogenic responses (3-fold increase of cell number). Proliferative effects of trypsin or AP2 were also observed in other cell lines expressing PAR-2. These data show that subnanomolar concentrations of trypsin, acting at PAR-2, promoted the proliferation of human colon cancer cells. The results of this study indicate that trypsin could be considered as a growth factor and unravel a new mechanism whereby serine proteases control colon tumours. © 2001 Cancer Research Campaign http://www.bjcancer.co

Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases.

We have examined protease-mediated activation of the mitogen-activated protein (MAP) kinase cascade in rat aortic smooth-muscle cells and bovine pulmonary arterial fibroblasts. Exposure of smooth-muscle cells to trypsin evoked rapid and transient activation of c-Raf-1, MAP kinase kinase 1 and 2 and MAP kinase that was sensitive to inhibition by soybean trypsin inhibitor. The actions of trypsin were closely mimicked by the proteinase-activated receptor 2 (PAR-2)-activating peptide sequence SLIGRL but not LSIGRL. Peak MAP kinase activation in response to both trypsin and SLIGRL was also dependent on concentration, with EC50 values of 12.1 +/- 3.4 nM and 62.5 +/- 4.5 microM respectively. Under conditions where MAP kinase activation by SLIGRL was completely desensitized by prior exposure of smooth-muscle cells to the peptide, trypsin-stimulated MAP kinase activity was markedly attenuated (78.9 +/- 15.1% desensitization), whereas the response to thrombin was only marginally affected (16.6 +/- 12.1% desensitization). Trypsin and SLIGRL also weakly stimulated the activation of the MAP kinase homologue p38 in smooth-muscle cells without any detectable activation of c-Jun N-terminal kinase. Strong activation of the MAP kinase cascade and modest activation of p38 by trypsin were also observed in fibroblasts, although in this cell type these effects were not mimicked by SLIGRL nor by the thrombin receptor-activating peptide SFLLRNPNDKYEPF. Reverse transcriptase-PCR analysis confirmed the presence of PAR-2 mRNA in smooth-muscle cells but not fibroblasts. Our results suggest that in vascular smooth-muscle cells, trypsin stimulates the activation of the MAP kinase cascade relatively selectively, in a manner consistent with an interaction with the recently described PAR-2. Activation of MAP kinase by trypsin in vascular fibroblasts, however, seems to be independent of PAR-2 and occurs by an undefined mechanism possibly involving novel receptor species

The assessment of left ventricular diastolic function: guidance and recommendations from the British Society of Echocardiography

Impairment of left ventricular (LV) diastolic function is common amongst those with left heart disease and is associated with significant morbidity. Given that, in simple terms, the ventricle can only eject the volume with which it fills and that approximately one half of hospitalisations for heart failure (HF) are in those with normal/'preserved' left ventricular ejection fraction (HFpEF) (Bianco et al. in JACC Cardiovasc Imaging. 13:258-271, 2020. 10.1016/j.jcmg.2018.12.035), where abnormalities of ventricular filling are the cause of symptoms, it is clear that the assessment of left ventricular diastolic function (LVDF) is crucial for understanding global cardiac function and for identifying the wider effects of disease processes. Invasive methods of measuring LV relaxation and filling pressures are considered the gold-standard for investigating diastolic function. However, the high temporal resolution of trans-thoracic echocardiography (TTE) with widely validated and reproducible measures available at the patient's bedside and without the need for invasive procedures involving ionising radiation have established echocardiography as the primary imaging modality. The comprehensive assessment of LVDF is therefore a fundamental element of the standard TTE (Robinson et al. in Echo Res Pract7:G59-G93, 2020. 10.1530/ERP-20-0026). However, the echocardiographic assessment of diastolic function is complex. In the broadest and most basic terms, ventricular diastole comprises an early filling phase when blood is drawn, by suction, into the ventricle as it rapidly recoils and lengthens following the preceding systolic contraction and shortening. This is followed in late diastole by distension of the compliant LV when atrial contraction actively contributes to ventricular filling. When LVDF is normal, ventricular filling is achieved at low pressure both at rest and during exertion. However, this basic description merely summarises the complex physiology that enables the diastolic process and defines it according to the mechanical method by which the ventricles fill, overlooking the myocardial function, properties of chamber compliance and pressure differentials that determine the capacity for LV filling. Unlike ventricular systolic function where single parameters are utilised to define myocardial performance (LV ejection fraction (LVEF) and Global Longitudinal Strain (GLS)), the assessment of diastolic function relies on the interpretation of multiple myocardial and blood-flow velocity parameters, along with left atrial (LA) size and function, in order to diagnose the presence and degree of impairment. The echocardiographic assessment of diastolic function is therefore multifaceted and complex, requiring an algorithmic approach that incorporates parameters of myocardial relaxation/recoil, chamber compliance and function under variable loading conditions and the intra-cavity pressures under which these processes occur. This guideline outlines a structured approach to the assessment of diastolic function and includes recommendations for the assessment of LV relaxation and filling pressures. Non-routine echocardiographic measures are described alongside guidance for application in specific circumstances. Provocative methods for revealing increased filling pressure on exertion are described and novel and emerging modalities considered. For rapid access to the core recommendations of the diastolic guideline, a quick-reference guide (additional file 1) accompanies the main guideline document. This describes in very brief detail the diastolic investigation in each patient group and includes all algorithms and core reference tables