Molecular alterations in fibroblasts exposed to Helicobacter pylori: a missing link in bacterial inflammation progressing into gastric carcinogenesis?

J Physiol Pharmacol. 2013 Feb;64(1):77-87.

Gracjana Krzysiek-Mączka, Aneta Targosz, Agata Ptak-Belowska, Edyta Korbut, Urszula Szczyrk, Malgorzata Strzalka, Tomasz Brzozowski

Department of Physiology, Jagiellonian University Medical College, Cracow, Poland. gracjana98@gmail.com

 

Abstract

Major human pathogen Helicobacter pylori (Hp) can colonize the gastric mucosa causing inflammation and being of potential risk for gastric cancer development but the contribution of fibroblasts to the pathogenesis of Hp in the stomach has been little studied. Normal stroma contains few fibroblasts, especially myofibroblasts, but their number rapidly increases in the reactive stroma surrounding inflammatory region and neoplastic tissue. We determined the effect of coincubation of cultured rat gastric fibroblasts with alive Hp on the transdifferentiation of fibroblasts into myofibroblasts associated with Hp-induced inflammation and neoplasia. Gastric mucosal samples were harvested from 8-week-old Spraque-Dowley rats and cultured to obtain the sub-confluent fibroblasts. The isolated fibroblasts were infected with 1 x 10(9) of live Hp (ATCC 700824, cagA+, vacA+) per dish and incubated in humidified atmosphere for 3, 24 and 48 hours. At respective times, fibroblasts were harvested and the expression of mRNA for a-smooth muscle actin (SMA), hypoxia inducible factor (HIF)-1a, collagen I, heat shock protein (HSP)-70, heme oxygenase (HO)-1, Bax and Ki67 transcripts was determined by RT-PCR with specific primers. Hp increased the transdifferentiation of fibroblasts into myofibroblasts as reflected by the time-dependent overexpression of mRNA for a-SMA. The increased expression of HIF-1a and collagen I was observed in fibroblasts co-cultured with Hp. The expression of HSP70 which was negligible in isolated fibroblasts incubated with vehicle (saline) showed time-dependent 2-3 fold increase in those incubated with Hp. The HO-1 mRNA was strongly expressed in rat gastric fibroblasts without or with the co-incubation with Hp. The mRNA for Bax was progressively downregulated within the time of incubation while no significant changes in expression of proliferation marker Ki67 were recorded. We conclude that Hp-induced transdifferentiation of fibroblasts into myofibroblasts involves an increased expression of the early carcinogenic marker HIF-1a, and inhibition of proapoptotic Bax expression, and 2) the overexpression of HSP70 and the unchanged expression HO-1 and Ki67 probably represent the enhanced protective activity of Hp-infected fibroblasts to maintain their own integrity under inflammatory action of this bacteria and its cytotoxins.

PMID: 23568974

 

Supplement:

The aim of our present study was to determine, whether gastric fibroblasts may represent cellular target of Hp affecting process of proliferation, apoptosis and cell defense system. We confirmed an increased differentiation  of fibroblasts into myofibroblasts via the enhancement of production of α-SMA mRNA (Fig. 1).

The enhanced production of  α-SMA correlated with the expression of mRNA for collagen type 1 (Fig. 2). That is in agreement with existing evidence showing that myofibroblasts positive for α-SMA correlate with enhanced production and deposition of extracellular matrix related proteins including collagen type I – an event associated with fibrosis (1, 2, 3) and gastric carcinoid tumours (4).

In cancer, changes in the stroma drive invasion and metastasis, the hallmarks of malignancy. The microenvironment is crucial for the mainteinance of cellular functions and tissue integrity. The development of neoplasia-induced change in the stroma may contribute to cancer invasion (5,6) and the appearance of myofibroblasts precedes the invasive stage of cancer. Experimental and clinical observations seem to suggest that myofibroblasts may be proinvasive and capable of producing proinvasive signals (5,6). For instance, α-SMA next to PDGFB-R is in fact regarded as cancer-associated fibroblasts (CAF) marker (3).

The enhanced upregulation of collagen I plays an important role in the impairment of tissue remodeling, angiogenesis and cancer invasion and metastasis caused by CagA-positive strain of Hp (7,8).

In our culture model system, the cagA and  vacA positive Hp strain upregulated in a time-dependent manner the expression of  HSP70 mRNA in fibroblasts (Fig. 3). This increase in HSP70 mRNA expression was observed already after 24 hours and subsequently noted at 48 h after exposure of cell culture to Hp. The expression of  Bax mRNA significantly decreased in fibroblasts already after 3 hours of their co-incubation with bacteria (Fig. 4). This findings  indicates that Hp inhibits apoptosis of fibroblasts in vitro.

At present, the mechanism by which Hp induces apoptosis in gastric cancer cells and interacts with HSP70 expression remains unknown. It has been postulated  that inactivation of HSP90 and HSP70 leads to loss of invasion in a variety of cancer cell types (9). Thus, we conclude that the influence of Hp on HSP70 may be cell type specific. In the case of fibroblasts this bug evidently promoted their differentiation with sustained rate of proliferation.

In our experimental model the HO-1 mRNA was also strongly expressed, but we observd no alteration in HO-1 expression level following coincubation with Hp (Fig. 5). Lakkisto et al. (10) showed that, HO-1 decreased accumulation and proliferation of fibroblasts, and down-regulated procollagen type I expression in the infarct area of heart failure model in rats. Lack of major alteration in HO-1 expression under our experimental settings is in agreement with the studies pointing to antifibrogenic properties of HO-1.

We conclude, that the exposure of fibroblasts to Hp promotes their differentiation into myofibroblasts, with possible excess of matrix production in vitro. The increased expression of HSP70  mRNA suggests that induction of HSP70 confers cytoprotection against Hp infection, by inhibiting the expression of Bax.

The Hp expressing CagA cytotoxin and also CagA by itself can induce ROS production via NADPH oxidase-dependent  pathway  (11) causing DNA damage, thus contributing to apoptosis in early stages of gastric carcinogenesis. However, this was not the case with respect to apoptosis in our study because Bax  expression was downregulated in fibroblasts incubated with Hp.

In culture of  MCF7 cancer cells and fibroblasts, the enhanced  ROS production and oxidative stress in adjacent fibroblasts as well as the activation of HIF-1α and  NFkB driven gene transcription were observed (12). According to literature  HIF-1α acts as a tumor promoter in stromal cells and  ROS are responsible for activation of  myofibroblast markers and extracellular matrix proteins associated with activated fibroblasts. Moreover, tumour hypoxia is now  recognised as a key factor driving  the development of malignancy, and  the master regulatory protein in the response of cells to change in oxygen levels via HIF-1α (12). We postulate here that HIF-1α upregulation (Fig. 6) could  represent one of  the prerequisites to development of gastric cancer development in  Hp infected  mucosa. This is supported by the fact that HIF-1α expression was upregulated in fibroblast coincubated with Hp.

Fibroblasts seem to cooperate with epithelial component in the stomach  infected with Hp, that may facilitate inflammation and peptic ulcer formation with potential risk to develop gastric cancer. However, further studies in fibroblasts affected by Hp and analysis of factors influenced by this bug at the protein level as well as the effect of incubation with various strains of Hp on the complex fibroblast-epithelial cell interaction are warranted.

Gracjana Krzysiek-Mączka-1Fig. 1.  RT-PCR analysis of mRNA expression for a-SMA  in rat gastric fibroblasts at 3, 24 and 48 h of co-incubation with Hp strain CagA+, VacA+ (ATTC 700824) and the ratio of a-SMA over β -actin at respective times of co-incubation with this bacteria. Results are mean ±S.E.M. of 6 determinations. Asterisk indicates a significant (p<0.05) change as compared to the value obtained at 3 h and 24 h of incubation with Hp.

Gracjana Krzysiek-Mączka-2Fig. 2. RT-PCR analysis of mRNA expression for collagen I  in rat gastric fibroblast at 3, 24 and 48 h of co-incubation with Hp (ATTC 700824) and the ratio of collagen I over β-actin  at respective times of co-incubation of these cells with bacteria. Results are mean ± S.E.M. of 6 determinations. Asterisk indicates a significant change (p<0.05) above these recorded at 3 h and 24 h of co-incubation with Hp.

Gracjana Krzysiek-Mączka-3Fig. 3. RT-PCR analysis of mRNA expression for HSP70  in rat gastric fibroblast at 3, 24 and 48 h  of co-incubation with Hp (ATTC 700824) and the ratio of HSP70 over β-actin at respective times of co-incubation of these cells with Hp. Results are mean ±S.E.M. of 6 determinations. Asterisk indicates a significant change (p<0.05) as compared to the value obtained at 3 h of incubation with Hp.

Gracjana Krzysiek-Mączka-4Fig. 4. RT-PCR analysis of mRNA expression for Bax  in rat gastric fibroblast at 3, 24 and 48 h of co-incubation with Hp (ATTC 700824) and the ratio of  Bax over  β-actin  at respective times of co-incubation of  these cells with Hp. Results are mean ±S.E.M. of 6 determinations. Asterisk indicates a significant change (p<0.05) as compared to the value obtained in control cells. Asterisk and cross indicate a significant change (p<0.05) as compared to the value obtained at 24 h.

Gracjana Krzysiek-Mączka-5Fig. 5. RT-PCR analysis of mRNA expression for HO-1  in rat gastric fibroblast at 3, 24 and 48 h of co-incubation with Hp (ATTC 700824) and the ratio of HO-1 over β-actin at respective times of co-incubation of these cells with Hp. Results are mean ±S.E.M. of 6 determinations.

Gracjana Krzysiek-Mączka-6Fig. 6. RT-PCR analysis of mRNA expression for HIF-1α  in rat gastric fibroblast at 3, 24 and 48 h of co-incubation with Hp (ATTC 700824) and the ratio of HIF-1α over β-actin at respective times of co-incubation of these cells with Hp. Asterisk indicates a significant change (p<0.05) compared to the value obtained at 3 h of incubation with Hp. Results are mean ±S.E.M. of 6 determinations. Asterisk indicates a significant change (p<0.05) as compared to the value obtained in control cells.

Reference:

1. Lin A, Hokugo A, Nishimura I. Wound closure and wound management: a new therapeutic molecular target. Cell Adh Migr 2010; 4: 396-399.

2. Duffield JS, Lupher M, Thannickal VJ, Wynn TA. Host responses in tissue repair and fibrosis. Annu Rev Pathol 2013; 8: 241-276.

3. Kumar S, Weaver VM. Mechanics, malignancy, and metastasis: the force journey of a tumor cell. Cancer Metastasis Rev 2009; 28: 113-127.

4. Ishikawa T, Ando T, Obayashi H, et al. Helicobacter pylori isolated from a patient with Menetrier’s disease increases hepatocyte growth factor mRNA expression in gastric fibroblasts: comparison with Helicobacter pylori isolated from other gastric diseases. Dig Dis Sci 2008; 53: 1785-1791.

5. De Wever O, Westbroek W, Verloes A, et al. Critical role of N-cadherin in myofibroblast invasion and migration in vitro stimulated by colon-cancer-cell-derived TGF-beta or wounding. J Cell Sci. 2004 Sep 15;117(Pt 20):4691-703.

6. De Wever O, Nguyen QD, van Hoorde L, et al. Tenascin-C and SF/HGF produced by myofibroblasts in vitro provide convergent pro-invasive signals to humancolon cancer cells through RhoA and Rac. FASEB J 2004; 18: 1016-1018.

7. Tegtmeyer N, Wittelsberger R, Hartig R, Wessler S, Martinez-Quiles N, Backert S. Serine phosphorylation of cortactin controls focal adhesion kinase activity and cell scattering induced by Helicobacter pylori. Cell Host Microbe 2011; 9: 520-531.

8. Wiese M, Eljaszewicz A, Andryszczyk M, et al. Immunomodulatory effects of Lactobacillous plantarum and Helicobacter pylori CagA+ on the expression of selected superficial molecules on monocyte and lymphocyte and the synthesis of cytokines in whole blood culture. J Physiol Pharmacol 2012; 63: 217-224.

9. Sims JD, McCready J, Jay DG. Extracellular heat shock protein Hsp70 and Hsp90a assist in matrix metalloproteinase-2 activation and breast cancer cell migration and invasion. PLoS One 2011; 6: e18848.

10. Lakkisto P, Siren JM, Kyto V, et al. Heme oxygenase-1 induction protects the heart and modulates cellular and extracellular remodelling after myocardial infarction in rats. Exp Biol Med (Maywood) 2011; 236: 1437-1448.

11. Cha B, Lim JW, Kim KH, Kim H. 15-Deoxy-D12,14- prostaglandin J2 suppresses RANTES expression by inhibiting NADPH oxidase activation in Helicobacter pylori-infected gastric epithelial cells. J Physiol Pharmacol 2011; 62: 167-174.

12. Chiavarina B, Whitaker-Menezes D, Migneco G, et al. HIF-1 alpha functions as a tumor promoter in cancer associated fibroblasts, and as a tumor suppressor in breast cancer cells: autophagy drives compartment-specific oncogenesis. Cell Cycle 2010; 9: 3534-3551.

Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann