Expression Patterns of the CLCA Homologs

To date, the expression of CLCA family members has mainly been studied by mRNA detection approaches such as in situ hybridization, Northern blot hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR; reviewed by GRUBER et al. 2002). Immunodetection studies to reveal the location of the protein have to date only been performed for bCLCA1 and bCLCA2 (ELBLE et al. 1997). Based on the available mRNA data, the tissue distribution patterns of CLCA homologs are distinct yet overlapping within single species and between different species. CLCA family members are mainly expressed in secretory epithelia, with some homologs being expressed in vascular endothelia as well. There are family members with more restricted tissue expression versus others with a more universal expression. In summary, the emerging picture is that of a multigene family with highly tissue-specific members, similar to the ClC family of voltage-gated Cl^- channels (GRUBER et al.

2002; see B.2.3).

The first bovine CLCA homolog, bCLCA1, was detected exclusively in the trachea by RT-PCR analysis with negative results obtained from lung, liver, brain and renal

papilla (CUNNINGHAM et al. 1995). However, ELBLE and coworkers (1997) detected both bCLCA1 and bCLCA2 by Northern blot and RT-PCR analyses in lung parenchyma, and bCLCA2 expression was additionally found in vascular endothelia of the lung and spleen.

In situ hybridization revealed hCLCA1 mRNA expression exclusively in small and large intestinal basal crypt epithelia and goblet cells whereas it was undetectable in heart, brain, placenta, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, peripheral blood leukocytes, stomach, thyroid gland, spinal cord, lymph node, trachea, adrenal gland, bone marrow and lung using Northern blot hybridization (GRUBER et al. 1998 a). However, TODA and colleagues (2002) recently detected the homolog in respiratory goblet cells using in situ hybridization.

The second human homolog, hCLCA2, was detected in the trachea, mammary gland and lung by Northern blot and RT-PCR analyses (GRUBER et al. 1999). In addition, ABDEL-GHANY and coworkers (2001) detected the homolog by RT-PCR analysis in cultured human pulmonary endothelial cells. Curiously, this CLCA homolog was also detected in a RT-PCR study in the epithelium of the cornea (ITOH et al. 2000).

GRUBER and PAULI (1999 b) detected the hCLCA3 homolog in lung, trachea, spleen, thymus and mammary gland by RT-PCR analysis. Northern blot and ribonucleic acid (RNA) dot blot hybridization analyses revealed mRNA expression of the fourth human family member, hCLCA4, predominantly in the colon. Northern blot hybridization revealed additional expression of the homolog in the small intestine and stomach, in the trachea, in the urogenital organs (urinary bladder, uterus, prostate, testis), in the salivary and mammary glands and, somewhat unique among CLCA family members, in the brain (AGNEL et al. 1999).

The expression pattern of the first murine member, mCLCA1, has been studied extensively. Northern blot and in situ hybridizations as well as RT-PCR studies performed by different groups (GANDHI et al. 1998; GRUBER et al. 1998 b; ROMIO et al. 1999; ABDEL-GHANY et al. 2002) revealed wide distribution of the homolog in numerous epithelia and few other cell types. For example, epithelia expressing

mCLCA1 were found by in situ hybridization as well as RT-PCR studies in the trachea including submucosal glands, in the bronchi, intestine, mammary glands, tubular structures of the kidney, gall bladder, liver bile ducts, pancreatic and salivary gland acini, oviduct, uterus and epididymis (GRUBER et al. 1998 b). Furthermore, mCLCA1 was detected in dermal, esophageal and corneal basal keratinocytes, in the germinal centers of spleen and lymph nodes, in spermatids (GRUBER et al. 1998 b) and by Northern blot hybridization in the brain (GANDHI et al. 1998). Vascular endothelia in the lung also expressed the homolog (ABDEL-GHANY et al. 2002).

Northern blot expression analysis of the second murine CLCA homolog, mCLCA2, has only been performed in mammary gland and lung (LEE et al. 1999). The results revealed expression of the homolog predominantly in the involuting mammary gland and at low levels in the lung. RT-PCR studies confirmed restricted mCLCA2 expression during early involution of the mammary gland (LEE et al. 1999). However, the extremely close sequence proximity between mCLCA1 and mCLCA2 (96 % cDNA identity; see B.3.1, Table B2) demanded careful reconsideration of the distribution patterns obtained. Cross-hybridization could not be excluded in particular for the mCLCA1 probes used in the studies prior to the discovery of mCLCA2. A real-time RT-quantitative PCR analysis based on gene-specific primers revealed the differential mCLCA1 and mCLCA2 expression patterns in a broad tissue spectrum (HORSTMEIER, in press). Interestingly, widely overlapping expression of both homologs was found in almost all tissues analyzed including trachea and intestines.

Moreover, as the previous in situ hybridization signals had been obtained from only a single cell type within each tissue (GRUBER et al. 1998 b), these data suggest that both homologs are expressed by the same cells, e.g., by enterocytes in the small and large intestines. In contrast, virtually only mCLCA1 was selectively expressed in the lung, spleen, bone marrow, lymph nodes, liver and aorta, and mCLCA2 was the predominantly expressed homolog in the lactating and involuting mammary glands, in the thymus and in the epididymis. For the first time, CLCA expression was also detected in fetal lung, liver, kidney and intestine for both mCLCA1 and mCLCA2.

Recently, two RT-PCR studies based on non-discriminating mCLCA1/2 primers revealed expression of one or both murine homologs in aortic endothelial cells

(PAPASSOTIRIOU et al. 2001) and expression of mCLCA1, as confirmed by subsequent sequencing, in vascular smooth muscle cells (BRITTON et al. 2002). The third murine CLCA family member, mCLCA3, was detected by in situ hybridization in goblet cells of the small and large intestines whereas Northern blot analysis revealed additional expression in the stomach, uterus and trachea but not in the liver, spleen, brain, skeletal muscle, skin, testis and salivary gland (KOMIYA et al. 1999). In situ hybridization and RT-PCR analyses revealed expression of mCLCA4 in mucosa- and serosa-free intestines, in the stomach, uterus, gall bladder and endothelia of aorta and lung vessels (ELBLE et al. 2002). However, cross-hybridization of the probes with the close relatives mCLCA1 and mCLCA2 (85 % cDNA identity to mCLCA4; see B.3.1, Table B2) cannot be excluded.

The only known porcine CLCA homolog to date, pCLCA1, was detected in villus and crypt epithelia of the ileum by RT-PCR. The signal was predominantly associated with isolated epithelial cells scattered throughout the villus, consistent with the location of goblet cells (GASPAR et al. 2000). In situ hybridization and RT-PCR analyses further revealed pCLCA1 expression in the trachea including submucosal glands but not in the large intestine or stomach (GASPAR et al. 2000).

In summary, the heterogeneity in their tissue expression patterns suggests differences in the individual physiological roles among the various CLCA family members. It suggests that in the course of mammalian evolution, certain CLCA homologs may have acquired tissue-specific functions that even differ within single-and between distinct species.