THEME
The Future of GI and Liver Research: Editorial Perspectives
I. Visions of epithelial research

Marshall H. Montrose

Indiana University School of Medicine, Department of Cellular and Integrative Physiology, Indianapolis, Indiana 46202


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EPITHELIAL PROTEINS
EPITHELIAL CELL CONSORTIUM
HOT TOPICS IN EPITHELIAL...
REFERENCES

Epithelial cells are gatekeepers that sit at the interface between two compartments. By controlling the flow of molecules and information between two compartments, epithelial cells provide unique benefit to the body. This article provides a brief appraisal of our current knowledge about the functions of gastrointestinal epithelial cells as a functionally diverse set of cells mediating transepithelial transport and as a continually renewing layer of cells. The convergence of new methodologies in laser capture microdissection, microarray analyses, microscopic analyses, and generation of mutant animals provides an exciting template for future research.

microscopy; genomics; proteomics; stem cell; ion transport; gastrointestinal


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EPITHELIAL PROTEINS
EPITHELIAL CELL CONSORTIUM
HOT TOPICS IN EPITHELIAL...
REFERENCES

EPITHELIAL CELLS REALLY HAVE only two functions. One is to block undesirable materials from crossing the epithelial layer, and the other is to selectively transfer desirable information or materials between the two compartments that the epithelial layer faces. Both functions are the result of cellular specialization and a fascinating constellation of tissue-specific proteins. Here, I will discuss how the convergence of technological innovations is poised to help resolve some physiological questions about epithelial function. Examples are drawn mostly from the literature on ion transport function in the gastrointestinal tract.


    EPITHELIAL PROTEINS
TOP
ABSTRACT
INTRODUCTION
EPITHELIAL PROTEINS
EPITHELIAL CELL CONSORTIUM
HOT TOPICS IN EPITHELIAL...
REFERENCES

Study of individual epithelial proteins has reached a pinnacle. Discovery and dissection of proteins have accelerated to an astounding pace. For example, many of the relevant transport proteins that mediate transepithelial salt absorption, solute absorption, and salt secretion have been identified and cloned during the past decade. Due to homology cloning, molecules that are likely transporters are now being discovered faster than their physiological role can be defined. A wonderful example is the putative anion transport (PAT), proteins in the SLC26A family, related to the DRA/CLD protein that causes congential chloride diarrhea (14). PAT family members are present in numerous tissues including the stomach, intestines, and pancreas, but as yet, only hints and allegations about their physiological function have been possible (27). The continuing discovery of new proteins should give us pause in the certainty that we truly know which molecules are mediating the physiological events we hold near and dear.

For the physiologist and molecular geneticist, inherited disorders provide a relevant and exciting template on which a physiological role for individual proteins can be tested. For example, it is widely accepted that the Na+/H+ exchanger (NHE) isoform NHE3 is the apical exchanger responsible for electroneutral sodium absorption in the small intestine (25). However, the complete lack of apical Na+/H+ exchange in congential sodium diarrhea cannot be explained by mutations in either NHE3 or NHE2, the second apical isoform known to be present in the small intestinal epithelium (4, 17). Maybe we have new NHE families yet to discover or a mandatory accessory protein of NHE3 is the target of mutation. Independent of explanation, the known NHE isoforms are clearly not uniquely required to elicit an apical Na+/H+ exchange function. Even in cases in which the relevant protein is firmly locked into a physiological role, the debates do not cease. Thus, although no one would argue that the CFTR is absolutely necessary for fluid and ion secretion (22), CFTR is probably the most notorious case of a protein that defies precise functional definition. Arguments still abound concerning the relative importance of CFTR to mediate chloride fluxes and bicarbonate fluxes as well as its ability to regulate the function of other proteins. Thanks to nature's endless versatility and inventiveness, developing a full understanding of even an individual membrane protein may take more than a decade, and we have numerous new candidates waiting to be discovered and examined. Fortunately, multifactorial diseases with an inherited component such as Crohn's disease, colon cancer, and diabetes are becoming tractable with modern genetic tools and in this way are becoming active contributors to our knowledge base of genes and proteins crucial to physiological processes.

Despite the challenges still faced in analysis of individual proteins, we are advancing to the next stage in studies of individual intestinal epithelial cells in native tissue.


    EPITHELIAL CELL CONSORTIUM
TOP
ABSTRACT
INTRODUCTION
EPITHELIAL PROTEINS
EPITHELIAL CELL CONSORTIUM
HOT TOPICS IN EPITHELIAL...
REFERENCES

When epithelial cells are involved, it never seems enough to have just one differentiated cell type. Diversity is the rule and not the exception, so epithelial function in a tissue is usually the result of combining the talents of multiple epithelial cell types. The major classes of epithelial cell types in any tissue have often been defined based largely on their morphological characteristics and/or cellular localization. Increasingly, this has been shown to be an overly conservative definition. We are entering the era in which cells may be more precisely defined by their individual protein composition or the constellation of functions individual cells perform in situ.

As we improve our ability to characterize single cells, we will be able to resolve whether there is a smoother spectrum of cellular function compared with the classically defined epithelial cell types. It will be fascinating to see what this means in terms of mixing epithelial functions that are thought of as residing in separate cells. Such work has already begun to expand our understanding of "absorptive" and "secretory" cells in the gastrointestinal tract. One of the paradigm shifts over the past decade has been in the realignment of our knowledge about colonic crypts. Classically believed to be a purely secretory structure, evidence has now unequivocally shown that crypts can be fluid absorptive. Originally hypothesized by Richard Naftalin (18), strong evidence for sodium-dependent crypt fluid absorption comes from direct measurements of fluid movements in isolated crypts (9, 24). The crypt absorptive process is likely to be electroneutral not electrogenic (13), but otherwise the molecular identity of the absorptive transporters remains uncertain. It does seem clear that apical Na+/H+ exchange, a function classically assigned to sodium absorptive cells, is also found deep in crypts (5, 21).

Because it is firmly established that crypts are fluid secretory, a substantial mystery lingers concerning how the same crypts can mediate both secretion and absorption (9, 24). Are both functions performed simultaneously? Do individual cells perform both these functions, or are neighboring cells dividing up these tasks? Do mucin secretory or enteroendocrine cells partake in these ion transport functions (15)? Two methods seemed poised to address such questions. One is the use of laser-capture microdissection in combination with gene-expression profiling, and the second is microscopic analysis of individual cell function. Ideally, both can be used to characterize the individual cell types that exist in defined locations of epithelial layers.

Laser-capture microdissection offers the promise of literally plucking a cell of interest from a tissue for detailed analyses. At present, the technology for grabbing cells (using a laser to stick cells to a plastic substrate) requires that the cells of interest be directly accessible at the surface of the sample and be stable (i.e., dead). The current state of the art is that portions of small intestinal crypts have been excised from tissue sections, with an RNA/DNA analysis possible from an aggregate of ~100 crypts (~2,000 cells) (29). Use of ~10,000 intestinal cells has been reported to be sufficient for quantitative microarray analysis after amplification of cDNAs (12). Thus gene-expression profiling of intestinal epithelial cells has started in earnest but not yet with the ability to analyze individual cells. Fortunately, microarray analysis is poised to report from individual cells (at least at the mRNA level), so we shall soon be able to characterize individual epithelial cells in a way never possible before (10, 26). The improving sensitivity of proteomics analysis is remarkable, but is still orders of magnitude away from single-cell analyses of protein composition (28).

Advanced microscopy methods offer the complementary promise of analyzing the function of individual living cells within intact tissue structures. Much of this promise is already available through the use of confocal microscopy to study living cells either in isolated intestinal tissues or in vivo preparations using fluorescent reporters (5, 7). However, because confocal imaging is degraded strongly when focusing deep into tissue specimens (6), there is widespread anticipation that two-photon microscopy will become the standard tool for deep-tissue imaging. Two-photon microscopy excites fluorescence using two low-energy photons instead of the usual one high-energy photon (8). This difference in the physics of excitation provides the remarkable property that two-photon fluorescence comes from only a localized region in the sample, and thereby two-photon imaging excels at removing haze (unwanted fluorescence) when imaging deep in light-scattering native tissues. Although fluorescence microscopy methods have classically imaged synthetic fluorescent chemicals, genetically encoded fluorescent proteins are rapidly ascending to become a standard method for not only localizing proteins, but for analyzing protein-protein interactions and protein function (30). The increasing incorporation of such protein reporters into transgenic animals will be of crucial importance in the coming years.

Mutant animals can now be sporting transgenes that are inducible, tissue-specific, and/or reversible. These wonderful technologies are matched only by those able to now eliminate genes (knockouts) via mechanisms that can be conditional (induced at a specific time/place) or reversible. An excellent article (2) has recently reviewed such technologies, so they will not be detailed here. I will simply note that our ability to manipulate and analyze the cellular environment of native tissue will soon be comparable (or superior) to what we currently perform in tissue culture models.


    HOT TOPICS IN EPITHELIAL DIVERSITY
TOP
ABSTRACT
INTRODUCTION
EPITHELIAL PROTEINS
EPITHELIAL CELL CONSORTIUM
HOT TOPICS IN EPITHELIAL...
REFERENCES

Given the emerging technologies for analyzing individual cell function, there are some fascinating areas to explore that have not previously been attainable.

One emerging area of cell-specific function that will be gaining attention is organellar diversity. Historically, examination of epithelial function and regulation has focused on alterations in plasma membrane or cytosolic proteins. This ignores an entire intracellular domain in which cells can further distinguish themselves from neighbors. Organellar diversity is more than just the presence or absence of specific structures (e.g., mucin granules); it can be subtle adjustments to ubiquitous organelles. For instance, there are reports of cell-type and tissue-specific expression of mitochondrial transporters (16), which may sensitize mitochondria to specific carbon sources. Similarly, variable expression of mitochondrial proteins sensitizes certain cell types to defined routes of apoptosis (20). There is strong evidence for redundant function between two Golgi copper-ATPase (ATP7) transporters (1, 19) that nonetheless can lead to two diseases with different tissue involvement (Menkes or Wilson disease) when the relevant genes are mutated (23). Through recent advances in designing GFP-based reporters of cellular function that can be targeted to organelles (30), we now have the tools to resolve differences in organellar function between individual cells and gain a broader view of cellular diversity.

A second area for advanced exploration is concerning the elusive tissue stem cells. In both the liver and intestine, epithelial cells have the capacity to self-renew. In the liver, this occurs in response to partial hepatectomy and other damaging insults, but in the intestine, this is a normal daily event, because each epithelial cell only has a lifetime of ~1 wk.

Intestinal epithelial stem cells are the progenitor of all cells in the epithelium and reside near the base of small intestinal crypts (directly above Paneth cells). Despite these sure statements, intestinal stem cells seem almost surreal. They are inferred from the ability of the intestinal epithelium to regenerate. They are counted indirectly from the kinetics of daughter cell replication, death, and migration. Their position is extrapolated from the migration speed of their daughter cells. In summary, their existence is indisputable from the vast weight of the evidence, yet they are never observed directly (3). As has been argued by Kaeffer (11), it seems unlikely that we will be able to pick out a stem cell by the expression of a single protein. Rather, we will need the power of microarrays to define expression profiles of a variety of biomarkers to identify the stem cell by a unique signature pattern vs. neighboring cells. If we are lucky, maybe the power of the new microscopy methods can also resolve unique functions of stem cells that will allow them to clearly stand out from neighboring cells.

In summary, in the pregenomic era, reductionist approaches helped isolate specific proteins and functions that were keys to tissue function. Now it is common to know protein sequences but to have protein functions remains speculative. Reductionist model systems will continue to help us to more completely assign protein function. As we move deeper into the postgenomic era, attention will be turning toward exploring specific protein function in the more complex models of native tissue. With the use of normal and mutant animal models, integrative approaches must explore how (and where) specific proteins function in native tissues and cope with interactions among multiple molecules in the tissue milieu. Our responsibility and role as physiologists is to place observations of cellular and molecular processes firmly in the context of tissue, organ, and body function. Integrative biology is the challenge, and the tools have finally arrived.


    ACKNOWLEDGEMENTS

In an attempt at brevity, this article fails to quote the work of numerous investigators who have made outstanding contributions in the areas discussed as well as neglecting numerous other exciting topics of epithelial research. The forbearance of the audience is requested.


    FOOTNOTES

Address for reprint requests and other correspondence: M. H. Montrose, Indiana Univ. School of Medicine, Dept. of Cellular and Integrative Physiology, Med Sci 307, 635 Barnhill Dr., Indianapolis, IN 46202-5120 (E-mail: mmontros{at}iupui.edu).

10.1152/ajpgi.00547.2002


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EPITHELIAL PROTEINS
EPITHELIAL CELL CONSORTIUM
HOT TOPICS IN EPITHELIAL...
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 284(4):G547-G550
0193-1857/03 $5.00 Copyright © 2003 the American Physiological Society




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