Cell-cell adhesion and RhoA-mediated actin polymerization are independent phenomena in microtubule disrupted keratinocytes.

E-cadherin-mediated adherens junction formation and maintenance are thought to involve actin filament rearrangements through the action of small GTPases. Recently, we demonstrated that microtubule disruption in normal human epidermal keratinocytes grown in low calcium media conditions induces cell-cell adhesion by redistribution of endogenous E-cadherin, and it promotes stress fiber formation. This actin rearrangement was apparently mediated by RhoA activation. This model system therefore provides a tool with which to dissect relationships between cell-cell adhesion and Rho-mediated stress fiber formation. In this study, we have demonstrated in normal human epidermal keratinocytes that disruption of actin structures including stress fibers does not interfere with E-cadherin redistribution during microtubule-induced cell-cell adhesion. Moreover, this cell-cell adhesion could not be blocked by RhoA inactivation at the level for inhibition of stress fiber formation. Additionally, in the immortalized HaCaT keratinocyte cell line, which does not undergo cell-cell adhesion after microtubule disruption in low calcium conditions, expression of dominant-active RhoA could induce stress fiber formation without inducing adhesion. On the other hand, a variant of the HaCaT cell line, HC-R1, showed microtubule-disruption-induced cell-cell adhesion without stress fiber formation. Together, our results suggest that, in keratinocytes, the process of cell adhesion can occur independently of RhoA-mediated stress fiber formation.

High-throughput investigation of osteoblast response to polymer crystallinity: influence of nanometer-scale roughness on proliferation.

A high-throughput method for analyzing cellular response to crystallinity in a polymer material is presented. Variations in crystallinity lead to changes in surface roughness on nanometer length scales, and it is shown that cells are exquisitely sensitive to these changes. Gradients of polymer crystallinity were fabricated on films of poly(L-lactic acid) using a gradient in annealing temperature. The resultant morphologies were characterized using an atomic force microscope. Root-mean-square (rms) roughness values ranging from 0.5 to 13 nm were created on a single sample. MC3T3-E1 osteoblastic cells were cultured for 1, 3 and 5 d, and the number of cells was measured using automated fluorescence microscopy. It is shown that the rate of proliferation on the smooth regions of the films is much greater than that on the rough regions, and a monotonic variation in rate is observed as a function of roughness. The critical rms roughness, above which a statistically significant reduction in rate of proliferation occurs, was approximately 1.1 nm. Fluorescence microscopy measurements on immunostained cells indicate there is no significant change in cell area, the number or type of adhesions formed, or the degree of actin polymerization. Results from enzyme-linked immunofluorescence assays indicated that there was no detectable change in adhesion protein accessibility, suggesting the cells directly respond to substrate topography. The use of the gradient library approach yielded the functional dependence of cell proliferation on nanometer-scale roughness and gave a sensitive estimate of the critical roughness for which a decrease in proliferation is observed.

Cell-matrix adhesions on poly(vinyl alcohol) hydrogels.

Cell-matrix adhesions regulate cell morphology, intracellular signaling, gene expression, and phenotype. Understanding how different methods of attaching matrix proteins to substrates affect the molecular arrangement of these adhesions offers the possibility of controlling cell function and architecture. The goal of this study was to visualize and quantify the cell-matrix adhesions formed by human fibroblasts on the matrix protein fibronectin covalently attached to poly(vinyl) alcohol (PVA) hydrogels. These adhesions were then compared with the cell adhesions formed in routine cell culture on fibronectin noncovalently coated onto glass coverslips or those formed on fibronectin covalently immobilized onto glass coverslips. Cell adhesions were characterized by immunofluorescence confocal microscopy utilizing paxillin as a marker for focal adhesions and alpha(5) integrin as a marker for fibrillar adhesions. As expected, distinct focal and fibrillar adhesions were observed in routine cell culture on coverslips coated noncovalently with fibronectin. Cells cultured on fibronectin covalently linked to PVA demonstrated diminished spatial separation of paxillin and alpha(5) integrin, accompanied by a reduction in fibrillar adhesions and fibronectin fibrillogenesis. Cells on fibronectin covalently immobilized on glass displayed the strongest marker colocalization and the most complete loss of fibrillar adhesions and lack of fibrillogenesis. These results indicate that fibronectin-conjugated PVA promotes the formation of cell adhesion structures intermediate in composition between those formed on noncovalently attached and covalently immobilized fibronectin. Furthermore, they imply that bioactive polymers can selectively induce specific cell-matrix adhesions, a characteristic that may have consequences in various tissue-engineering applications.

Systems analysis of salivary gland development and disease.

Branching morphogenesis is a crucial developmental process in which vertebrate organs generate extensive epithelial surface area while retaining a compact size. In the vertebrate submandibular salivary gland, branching morphogenesis is crucial for the generation of the large surface area necessary to produce sufficient saliva. However, in many salivary gland diseases, saliva-producing acinar cells are destroyed, resulting in dry mouth and secondary health conditions. Systems-based approaches can provide insights into understanding salivary gland development, function, and disease. The traditional approach to understanding these processes is the identification of molecular signals using reductionist approaches; we review current progress with such methods in understanding salivary gland development. Taking a more global approach, multiple groups are currently profiling the transcriptome, the proteome, and other 'omes' in both developing mouse tissues and in human patient samples. Computational methods have been successful in deciphering large data sets, and mathematical models are starting to make predictions regarding the contribution of molecules to the physical processes of morphogenesis and cellular function. A challenge for the future will be to establish comprehensive, publicly accessible salivary gland databases spanning the full range of genes and proteins; plans are underway to provide these resources to researchers in centralized repositories. The greatest challenge for the future will be to develop realistic models that integrate multiple types of data to both describe and predict embryonic development and disease pathogenesis.

The Krüppel-like factor epiprofin is expressed by epithelium of developing teeth, hair follicles, and limb buds and promotes cell proliferation.

We identified a cDNA clone for epiprofin, which is preferentially expressed in teeth, by differential hybridization using DNA microarrays from an embryonic day 19.5 mouse molar cDNA library. Sequence analysis revealed that this cDNA encodes a member of the Krüppel-like factor family containing three characteristic C2H2-type zinc finger motifs. The full-length cDNA was obtained by the 5' Cap capture method. Except for its 5'-terminal sequence, the epiprofin mRNA sequence is almost identical to the predicted sequence of Krüppel-like factor 14/Sp6 (specificity protein 6), which was previously identified in expressed sequence tag data bases and GenBank by an Sp1 zinc finger DNA-binding domain search (Scohy, S., Gabant, P., Van Reeth, T., Hertveldt, V., Dreze, P. L., Van Vooren, P., Riviere, M., Szpirer, J., and Szpirer, C. (2000) Genomics 70, 93-101). This sequence difference is due to differences in the assignment of the location of exon 1. In situ hybridization revealed that epiprofin mRNA is expressed by proliferating dental epithelium, differentiated odontoblast, and also hair follicle matrix epithelium. In addition, whole mount in situ hybridization showed transient expression of epiprofin mRNA in cells of the apical ectodermal ridge in developing limbs and the posterior neuropore. Transfection of an epiprofin expression vector revealed that this molecule is localized in the nucleus and promotes cell proliferation. Thus, epiprofin is a highly cell- and tissue-specific nuclear protein expressed primarily by proliferating epithelial cells of teeth, hair follicles, and limbs that may function in the development of these tissues by regulating cell growth.