Induction of Protein Tyrosine Kinase 6 in Mouse Intestinal Crypt Epithelial Cells Promotes DNA Damage–Induced Apoptosis

doi:10.1053/j.gastro.2009.05.054

Gastroenterology

Volume 137, Issue 3, September 2009, Pages 945-954

https://doi.org/10.1053/j.gastro.2009.05.054 Get rights and content

Background & Aims

Protein tyrosine kinase 6 (PTK6) is expressed in epithelial linings of the gastrointestinal tract. PTK6 sensitizes the nontransformed Rat1a fibroblast cell line to apoptotic stimuli. The aim of this study was to determine if PTK6 regulates apoptosis in vivo after DNA damage in the small intestine.

Methods

Wild-type and Ptk6^−/− mice were subjected to γ-irradiation; intestinal tissues were collected, protein was isolated, and samples were fixed for immunohistochemical analyses at 0, 6, and 72 hours after the mice were irradiated. Expression of PTK6 was examined in the small intestine before and after irradiation. Apoptosis and proliferation were compared between wild-type and Ptk6^−/− mice. Expression and activation of prosurvival signaling proteins were assessed.

Results

Irradiation induced PTK6 in crypt epithelial cells of the small intestine in wild-type mice. Induction of PTK6 corresponded with DNA damage–induced apoptosis in the wild-type small intestine. Following irradiation, the apoptotic response was impaired in the intestinal crypts of Ptk6^−/− mice. Increased activation of AKT and extracellular signal–regulated kinase (ERK)1/2 and increased inhibitory phosphorylation of the proapoptotic protein BAD were detected in Ptk6^−/− mice after irradiation. In response to the induction of apoptosis, compensatory proliferation increased in the small intestines of wild-type mice but not in Ptk6^−/− mice at 6 hours after irradiation.

Conclusions

PTK6 is a stress-induced kinase that promotes apoptosis by inhibiting prosurvival signaling. After DNA damage, induction of PTK6 is required for efficient apoptosis and inhibition of AKT and ERK1/2.

Section snippets

Animals and Tissues

Ptk6-null mice (B6.129SV-Ptk6^tm1Aty) have been previously described.¹³ Sex- and age-matched male mice at 8–10 weeks of age from heterozygous Ptk6^+/− breeding pairs were used. Samples from multiple animals of the same genotype are included in each figure. Whole-body irradiation was performed using a ¹³⁷Caesium γ-source (J. L. Shepherd model 6810; J. L. Shepherd, San Fernando, CA) at a dose of 8 Gy. At 0, 6, and 72 hours following irradiation, the mice were killed by co₂ anesthesia followed by

PTK6 Expression Is Induced in Intestinal Crypts After γ-Irradiation

To begin to assess the role of PTK6 following DNA damage, PTK6 protein levels were examined by immunoblotting in the small intestines of wild-type mice following total body γ-irradiation (8 Gy). A significant time-dependent increase in PTK6 protein levels was detected after irradiation (Figure 1A and B). PTK6 protein expression was localized in untreated and irradiated wild-type mice using immunohistochemistry. Consistent with prior reports,¹³ PTK6 protein expression was primarily restricted to

Discussion

Functions of PTK6 are not well understood and often seem paradoxical. A variety of data indicate that this intracellular tyrosine kinase has distinct activities in different cell types. Although PTK6 expression is associated with differentiation in the gastrointestinal tract and skin,3, 5, 6, 13 it is expressed at relatively high levels in a high percentage of human breast tumors but not in the normal mammary gland.⁹ In the normal prostate, PTK6 is localized to nuclei of epithelial cells but is

Acknowledgments

A.H.'s current address is: Bayer Schering Pharma AG, GDD, TCR in vivo 3, Muellerstr. 178, 13353 Berlin, Germany.

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PTK6 expression is induced by a variety of cellular stresses including DNA damage [49,50], hypoxia, and reactive oxygen species [45]. PTK6 expression is induced in the proliferating progenitor cell compartments of the intestine and skin after DNA-damage generated by different mechanisms, including gamma [49,50] and UV [51] irradiation and DNA-damaging drugs [50,52]. In cancer cells, PTK6 expression increased following treatment with radiation and chemotherapeutic agents, including doxorubicin [50], Taxol, 5′ fluorouracil [46], and gemcitabine [53].
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Protein tyrosine kinase 6 (PTK6, also called BRK) is an intracellular tyrosine kinase expressed in the epithelial linings of the gastrointestinal tract and the skin, where it is expressed in nondividing differentiated cells. We found that PTK6 expression increases in the epidermis following UVB treatment. To evaluate the roles of PTK6 in the skin following UVB-induced damage, we exposed back skin of Ptk6 +/+ and Ptk6 -/- SENCAR mice to incremental doses of UVB for 30 weeks. Wild-type mice were more sensitive to UVB and exhibited increased inflammation and greater activation of signal transducer and activator of transcription-3 (STAT3) than Ptk6-/- mice. Disruption of Ptk6 did not have an impact on proliferation, although PTK6 was expressed and activated in basal epithelial cells in wild-type mice following UVB treatment. However, wild-type mice exhibited shortened tumor latency and increased tumor load compared with Ptk6-/- mice, and STAT3 activation was increased in these tumors. PTK6 activation was detected in UVB-induced tumors, and this correlated with increased activating phosphorylation of focal adhesion kinase (FAK) and breast cancer anti-estrogen resistance 1 (BCAR1). Activation of PTK6 was also detected in human squamous cell carcinomas of the skin. Although PTK6 has roles in normal differentiation, it also contributes to UVB-induced injury and tumorigenesis in vivo.
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A subsequent study corroborated BRK expression specifically in the differentiating epithelial cells of the colon in the gastrointestinal (GI) tract [22]. However, later studies by the same group revealed the induction of BRK expression even in the proliferating epithelial cells of the crypt compartment besides the non-proliferating differentiated epithelial cells of the villi upon exposure to ionizing radiation [80]. Concordantly, it has been reported that under normal conditions BRK is primarily confined to the non-dividing differentiated intestinal cells but becomes induced in the proliferating crypt epithelial cells upon treatment with the carcinogen Azoxymethane (AOM) [81].
Twenty years have passed since the non-receptor tyrosine kinase, Breast tumor kinase (BRK) was cloned. While BRK is evolutionarily related to the Src family kinases it forms its own distinct sub-family referred here to as the BRK family kinases. The detection of BRK in over 60% of breast carcinomas two decades ago and more remarkably, its absence in the normal mammary gland attributed to its recognition as a mammary gland-specific potent oncogene and led BRK researchers on a wild chase to characterize the role of the enzyme in breast cancer. Where has this chase led us? An increasing number of studies have been focused on understanding the cellular roles of BRK in breast carcinoma and normal tissues. A majority of such studies have proposed an oncogenic function of BRK in breast cancers. Thus far, the vast evidence gathered highlights a regulatory role of BRK in critical cellular processes driving tumor formation such as cell proliferation, migration and metastasis. Functional characterization of BRK has identified several signaling proteins that work in concert with the enzyme to sustain such a malignant phenotype. As such targeting the non-receptor tyrosine kinase has been proposed as an attractive approach towards therapeutic intervention. Yet much remains to be explored about (a) the discrepant expression levels of BRK in cancer versus normal conditions, (b) the dependence on the enzymatic activity of BRK to promote oncogenesis and (c) an understanding of the normal physiological roles of the enzyme. This review outlines the advances made towards understanding the cellular and physiological roles of BRK, the mechanisms of action of the protein and its therapeutic significance, in the context of breast cancer.
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We identified protein tyrosine kinase 6 (PTK6) as a transcript that was significantly down-regulated in ESCC tissues and cell lines compared with nontumor tissues or immortalized normal esophageal cell lines. The promoter of the PTK6 gene was inactivated in ESCC tissues at least in part via hypermethylation and histone deacetylation. Knockdown of PTK6 in KYSE30 ESCC cells using small hairpin RNAs increased their ability to form foci, migrate, and invade extracellular matrix in culture and form tumors in nude mice. Overexpression of PTK6 in these cells reduced their proliferation in culture and tumor formation in mice. PTK6 reduced phosphorylation of Akt and glycogen synthase kinase (GSK)3β, leading to activation of β-catenin.
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However, a significant increase in apoptosis was detected in Ptk6+/+ mice compared with Ptk6−/− mice at the 6-hour time point after AOM treatment (Figure 3B and C). These data are consistent with prior reports that PTK6 expression plays an important role in DNA-damage−induced apoptosis in the small intestinal epithelium.10 Immunoblotting also revealed higher levels of cleaved caspase-3 at 6 hours post AOM injection in wild-type mice (Figure 3D).
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Ptk6^+/+ and Ptk6^−/− mice were injected with azoxymethane alone or in combination with dextran sodium sulfate; formation of aberrant crypt foci and colon tumors was examined. Effects of disruption of Ptk6 on proliferation, apoptosis, and STAT3 activation were examined by immunoblot and immunohistochemical analyses. Regulation of STAT3 activation was examined in the HCT116 colon cancer cell line and young adult mouse colon cells.
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: Conflicts of interest The authors disclose no conflicts.

: Funding Supported by National Institutes of Health grants DK44525 and DK068503 (to A.L.T.). A.H. received support from the German Academic Exchange Service (DAAD) and the Schering Foundation. J.J.G. is supported by an American Gastroenterological Association Foundation Graduate Student Research Fellowship Award and was supported by a National Research Service Award/National Institutes of Health institutional T32 training grant, “Training Program in Signal Transduction and Cellular Endocrinology” (T32 DK07739), from the National Institute of Diabetes and Digestive and Kidney Diseases.

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Basic—Alimentary TractInduction of Protein Tyrosine Kinase 6 in Mouse Intestinal Crypt Epithelial Cells Promotes DNA Damage–Induced Apoptosis

Background & Aims

Methods

Results

Conclusions

Section snippets

Animals and Tissues

PTK6 Expression Is Induced in Intestinal Crypts After γ-Irradiation

Discussion

Acknowledgments

Oral Oncol

Am J Pathol

J Biol Chem

Am J Pathol

J Biol Chem

Cell

Cell

Cell

J Biol Chem

Curr Biol

Cell

Cell Stem Cell

J Clin Invest

Brk, Srm, Frk, and Src42A form a distinct family of intracellular Src-like tyrosine kinases

Oncol Res

Tyrosine kinase gene expression in the mouse small intestine

Oncogene

A novel intracellular epithelial cell tyrosine kinase is expressed in the skin and gastrointestinal tract

Oncogene

BRK/Sik expression in the gastrointestinal tract and in colon tumors

Clin Cancer Res

A role for the epithelial-cell-specific tyrosine kinase Sik during keratinocyte differentiation

Proc Natl Acad Sci U S A

Role of breast tumour kinase in the in vitro differentiation of HaCaT cells

Br J Dermatol

Altered localization and activity of the intracellular tyrosine kinase BRK/Sik in prostate tumor cells

Oncogene

BRK tyrosine kinase expression in a high proportion of human breast carcinomas

Oncogene

The BRK tyrosine kinase is expressed in high-grade serous carcinoma of the ovary

Cancer Biol Ther

Protein tyrosine kinase 6 negatively regulates growth and promotes enterocyte differentiation in the small intestine

Mol Cell Biol

Basic—Alimentary Tract
Induction of Protein Tyrosine Kinase 6 in Mouse Intestinal Crypt Epithelial Cells Promotes DNA Damage–Induced Apoptosis