ARTICLE |
Correspondence to: Alfonso Baldi, Center for Experimental Research, Regina Elena Cancer Institute, Via delle Messi d'Oro, 156, 00158 Rome, Italy. E-mail: baldi@ifo.it
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Summary |
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The human HtrA family of proteases consists of three members: HtrA1, HtrA2, and HtrA3. In bacteria, the chief role of HtrA is recognition and degradation of misfolded proteins in the periplasm, combining a dual activity of chaperone and protease. In humans, the three HtrA homologues appear to be involved in diverse functions such as cell growth, apoptosis, allergic reactions, fertilization, control of blood pressure, and blood clotting. Previous studies using RNA blot hybridization have shown that the expression of HtrA1 is ubiquitous in normal human tissues. Here we show by immunohistochemistry (IHC) that HtrA1 is widely expressed, although different tissue distributions and/or levels of expression were detected in the different tissues examined. In particular, high to medium HtrA1 expression was detected in mature layers of epidermis, in secretory breast epithelium, in liver, and in kidney tubules of cortex, in concordance with its secretory properties. Furthermore, we show a higher protein expression level in the epithelium of proliferative endometrium, in contrast to epithelium of secretory endometrium, which is almost completely negative for this protein. This suggests a possible role for HtrA1 in the modulation of tissue activity in this organ. The various expression levels in human tissues indicate several possible roles for HtrA1 in different cell types. (J Histochem Cytochem 51:12791284, 2003)
Key Words: HtrA1, human tissues, immunohistochemistry, endometrium
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Introduction |
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The bacterial serine protease HtrA, also known as DegP, is a heat shock-induced, envelope-associated serine protease (
HtrA1 is the first identified member of the human HtrA protein family. It is a secreted protein, shown to be upregulated during osteoarthritis or to be significantly downregulated in transformed fibroblasts, in ovarian cancer, and in melanoma (
HtrA2, initially identified as a stress-activated protease, is considered to be a member of the apoptotic machinery that binds and therefore regulates the function of the inhibitors of apoptotic proteins (IAPs) (
In contrast to HtrA1 and HtrA2, the precise function of HtrA3 is still largely unknown. Given its higher homology with HtrA1, it is speculated that it could have similar functions. This hypothesis is strengthened by the observation that mRNAs coding for the two proteases display a similar RNA expression pattern in human tissues (
Here we describe a study of the expression pattern of HtrA1 in adult human tissues conducted by imunohistochemistry (IHC).
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Materials and Methods |
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Normal Tissues
Normal tissues from autopsies were obtained from the Section of Anatomic Pathology of the Second University of Naples, Italy. Tissues were formalin-fixed and paraffin-embedded. Representative sections of each specimen were stained with hematoxylineosin and were examined by a pathologist to confirm the histological preservation of the microanatomic structure. For each tissue examined, at least three specimens from two different individuals were analyzed. A human tissue array (AA4 Normal organs; Superbiochips, Korea), containing 59 normal human adult tissues, was also used.
Immunohistochemistry
IHC was carried out essentially as described previously (), very low (
), low (), moderate (), intense (), or a very high (). Noticeable quantitative variations in HrtA1 expression in staining intensity were observed, with less variation in the total number of stained cells.
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Results |
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We used IHC to determine the amount and localization of HtrA1 in a panel of different human tissues. HtrA1 was expressed widely, although a different tissue distribution and/or level of expression was detected in the different organs examined (Table 1). Noticeable quantitative variations in HrtA1 expression were observed in staining intensity, with less variation in the total number of stained cells.
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Expression of HtrA1 in Epithelial Tissues
Epithelial cells from either simple or stratified epithelium showed variable positive staining for HtrA1. Stratified epithelia, such as those from esophagus, cervix, and vagina, expressed a low level of this protein with a major localization in the mature and outer layers, with respect to basal layers that showed an undetectable level of the protein. In contrast, in the skin there was a high expression level for HtrA1 in mature layers of epidermis, whereas a low expression level was found in hair follicles, sebaceous glands, and sweat glands (Fig 1a). A medium expression level was seen in secretory breast epithelium compared to a low expression level in breast duct epithelium. Low immunoreactivity was detected in the stratified columnar epithelia of trachea, bronchi, and adjacent glands. Pneumocytes showed an undetectable level of the protein.
In the gastrointestinal system, low positive cytoplasmic staining for HtrA1 was found in secretory ducts, compared with an undetectable level in acini of salivary glands. An intense level was shown in the duct cells of the liver, whereas a low level was seen in the hepatocytes (Fig 1b). Low-level HtrA1 expression was seen in the stomach and gallbladder, and a moderate level was seen in the colon epithelia (Fig 1c). The exocrine portion of the pancreas showed a moderate level of expression for HtrA1, whereas the endocrine portion of the gland was less intense.
In the urinary system, HtrA1 was expressed at a medium level in all kidney tubules of cortex (Fig 1d), whereas a low expression of the protein was shown in the tubules of medulla and in the glomeruli. Uroepithelium and prostate epithelia were negative for HtrA1, whereas prostate stroma showed moderate positivity for this protein.
HtrA1 was expressed at a medium level in thyroid follicles. Its expression was low in the adrenal glands, where staining was more intense in the glomerulosa than in reticularis and fascicolata regions of the cortical portion, in contrast to an undetectable level of HtrA1 showed in the chromaffin portion of the glands.
The male reproductive system displayed low to undetectable positive cytoplasmic staining for HtrA1 in the epithelia. Low levels of expression were found in epididymis, in contrast with an undetectable level in testis and in epithelium of the seminal vesicle (Fig 1e), where only the stroma showed intermediate positivity for the protein.
Very interesting was the pattern of expression of HtrA1 in the female reproductive system. HtrA1 was expressed at an intermediate level, particularly in the epithelium of proliferating endometrium (Fig 1f), in contrast to epithelium of secretory endometrium, which was almost completely negative for this protein (Fig 1g). In the exocervix, an intermediate level of HtrA1 was found in the basal layer in contrast with the outer and mature layers, which showed a lower level of the protein. In the ovary, only the stroma showed an intermediate level of expression of HtrA1, whereas both oogonia and follicles showed a low level of the protein. Finally, greater expression of HtrA1 was found in third trimester placenta compared with placenta at the first trimester of gestation.
Expression of HtrA1 in Cardiovascular and Connective Tissues
Low immunoreactivity for HtrA1 was observed in skeletal muscle cells and in myocardial cells. An undetectable level was found in adipocytes, whereas a medium level was shown in chondrocytes, endothelial cells, and fibroblasts.
Expression of HtrA1 in Central and Peripheral Nervous System
All neurons from different areas of the brain, such as frontal cortex and midbrain and cells of the granular level of the cerebellum, displayed low staining for HtrA1. Perineural and endoneural cells of peripheral nerves and ganglion cells, such as astrocytes, oligodendroglial, and microglial cells of brain tissue, showed low levels of the protein.
Expression of HtrA1 in Hematopoietic Cells
An undetectable/low level of HtrA1 was observed in blood cells and in several lymphoid tissues.
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Discussion |
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In contrast to HtrA2, the precise function of HtrA1 is still largely unknown. It is reported to be a secreted protein (
An exhaustive knowledge of the differential cell- and tissue-specific pattern of expression of HtrA1 in normal human tissues is an essential requirement for a critical evaluation of the exact role played by this protein in cell homeostasis, especially in view of the fact that this serine protease is downmodulated in several human tumors, such as ovarian tumors (
We have shown a very particular expression of this serine protease in the endometrium. High protein expression is detected in the glandular epithelium of proliferating endometrium, in contrast to secretory endometrium that appeared almost completely negative for this protein. This peculiar pattern of expression of HtrA1 might account for distinct activities of this protein in this tissue. Intriguingly, it has been proposed that possible substrates of interest of HtrA1 include other proteases, extracellular matrix proteins, growth factors, and proteins that modulate growth factors (
In conclusion, our data concerning the IHC distribution of HtrA1 in normal human tissues demonstrate that the expression of this protease is modulated not only in pathological conditions but also in tissues with different physiological activities. Further studies aimed at investigating the transcription regulation of HtrA1 gene and at identifying functional targets of its protease activity will help to clarify the exact role of this protein in both physiological and pathological conditions.
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Footnotes |
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1 These authors contributed equally to this work.
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Acknowledgments |
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Supported in part by the AIRC and Ministero della Salute grants to M.G.P., by Futura-ONLUS and by Second University grants to A.B. We thank the International Society for the Study of Comparative Oncology for its continuous support.
We also thank Mr Giuseppe Falcone for his contribution to the image elaboration.
Received for publication March 10, 2003; accepted May 27, 2003.
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Literature Cited |
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Baldi A, Battista T, De Luca A, Santini D, Rossiello L, Baldi F, Natali PG et al. (2003) Identification of genes down-regulated during melanoma progression: a cDNA array study. Exp Dermatol 12:213-218[Medline]
Baldi A, De Luca A, Morini M, Battista T, Felsani A, Baldi F, Catricalà C et al. (2002) The HtrA1 serine protease is down-regulated during human melanoma progression and represses growth of metastatic melanoma cells. Oncogene 21:6684-6688[Medline]
Bakay M, Zhao P, Chen J, Hoffman EP (2002) A web-accessible complete transcriptosome of normal human and DMD muscle. Neuromusc Disord 12:S125-141[Medline]
Bulletti C, Jasonni VM, Polli V, Cappuccini F, Galassi A, Flamigni C (1991) Basement membrane in human endometrium: possible role of proteolytic enzymes in developing hyperplasia and carcinoma. Ann NY Acad Sci 622:376-382[Abstract]
Butzow R, Alfthan H, Julkunen M, Rutanen EM, Bohn H, Seppala M (1986) Human endometrium and menstrual fluid contain placental protein 5 (PP5). Hum Reprod 1:287-289[Abstract]
Clausen T, Southan C, Ehrmann M (2002) The HtrA family of proteases: implications for protein composition and cell fate. Mol Cell 10:443-455[Medline]
Faccio L, Fusco C, Chen A, Martinotti S, Bonventre JV, Zervos AS (2000) Characterization of a novel human serine protease that has extensive homology to bacterial heat shock endoprotease HtrA and is regulated by kidney ischemia. J Biol Chem 275:2581-2588
Gray CW, Ward RV, Karran E, Turconi S, Rowles A, Viglienghi D, Southan C et al. (2000) Characterization of human HtrA2, a novel serine protease involved in the mammalian cellular stress response. Eur J Biochem 267:5699-5710
Hettinger AM, Allen MR, Zhang BR, Goad DW, Malayer JR, Geisert RD (2001) Presence of the acute phase protein, bikunin, in the endometrium of gilts during estrous cycle and early pregnancy. Biol Reprod 65:507-513
Hu SI, Carozza M, Klein M, Nantermet P, Luk D, Crowl RM (1998) Human HtrA, an evolutionarily conserved serine protease identified as a differentially expressed gene product in osteoarthritic cartilage. J Biol Chem 273:34406-34412
Krojer T, GarridoFranco M, Huber R, Ehrmann M, Clausen T (2002) Crystal structure of DegP (HtrA) reveals a protease-chaperone machine. Nature 416:455-459[Medline]
Nagy P, Bisgaard HC, SantoniRugiu E, Thorgeirsson SS (1996) In vivo infusion of growth factors enhances the mitogenic response of rat hepatic ductal (oval) cells after administration of 2-acetylaminofluorene. Hepatology 23:71-79[Medline]
Nie GY, Hampton A, Li Y, Findlay JK, Salamonsen LA (2003a) Identification and cloning of two isoforms of human HtrA3, characterization of its genomic structure and comparison of its tissue distribution with HtrA1 And HtrA2. Biochem J 371:39-48[Medline]
Nie GY, Li Y, Minoura H, Batten L, Ooi GT, Findlay JK, Salamonsen LA (2003b) A novel serine protease of the mammalian HtrA family is up-regulated in mouse uterus coinciding with placentation. Mol Hum Reprod 9:279-290
Pallen MJ, Wren BW (1997) The HtrA family of serine proteases. Mol Microbiol 26:209-221[Medline]
Shirdar V, Sen A, Chien J, Staub J, Avula R, Kovats S, Lee J et al. (2002) Identification of underexpressed genes in early- and late-stage primary ovarian tumors by suppression subtraction hybridization. Cancer Res 62:262-270
Spiess C, Beil A, Ehrmann MA (1999) A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein. Cell 97:339-347[Medline]
van Loo G, van Gurp M, Depuydt B, Srinivasula SM, Rodriguez I, Alnemri ES, Gevaert K et al. (2002) The serine protease Omi/HtrA2 is released from mitochondria during apoptosis. Omi interacts with caspase-inhibitor XIAP and induces enhanced caspase activity. Cell Death Differ 9:20-26[Medline]
Verhagen AM, Silke J, Ekert PG, Pakusch M, Kaufmann H, Connolly LM, Day CL et al. (2002) HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins. J Biol Chem 277:445-454
Zumbrunn J, Trueb B (1996) Primary structure of a putative serine protease specific for IGF- binding proteins. FEBS Lett 398:187-192[Medline]