David Boettiger, Ph.D.

Professor Emeritus

boettige@mail.med.upenn.edu

RECENT PUBLICATIONS

RESEARCH SUMMARY

Cells receive signals from both their chemical and physical environment. The chemical microenvironment includes nutritional and hormonal factors and can be easily manipulated using tissue culture model systems. The physical environment includes the architecture of these microenvironments and the physical stresses which are exerted. The physical environment may be sensed through the receptors which are used to adhere cells to the interstitial extracellular matrix and to other cells. The integrin family of cell receptors are important contributors to these interactions. In addition to their functions in anchoring cells, they have been implicated in both the transfer of information from the cellular environment to elicit a physiological response and in transferring information about the state of the cell to the environment. These functions have been called outside-in and inside-out signaling.

Our laboratory has two goals: (1) to determine how the physical component of the signal is interpreted by the cell, and (2) to determine the role of these signals in cell cycle progression and in cell differentiation. It is our hypothesis that these signals are often interrupted by neoplastic transformation and that oncogenes, including src, target these pathways. The first problem is to build cell culture models which can modulate the physical environment and to devise methods for analysis of the physical forces which cells apply and which cells can withstand. We have used a combination of materials science, mechanical- and bio-engineering to supplement our approach these problems.

Our simplified model system focuses on a5b1 integrin which serves as a major receptor for fibronectin in the extracellular matrix. It is the interface between a5b1 and fibronectin which forms the cell's adhesive contact. Fibronectin in turn adsorbs or binds to a variety of both biological and inert surfaces and these surfaces can modulate the conformation of fibronectin in the binding. This modified fibronectin in turn interacts differently with a5b1. For example, interaction of fibronectin with unmodified polystyrene (as is commonly used for bacterial agar plates) results in a conformation of fibronectin which is about 1/10 as effective in cell binding compared to polystyrene modified for tissue culture (standard tissue culture plastic) and this is the reason that cells generally fail to grow on bacterial dishes. This can also be important in materials used for dental and orthopedic implants as we have shown recently for bioactive ceramics. For the analysis of strength of adhesion, we have developed a spinning disc device which is capable of exposing cells to a graded shear force which is sufficient to detach even well adherent cells. The strength of adhesion measured is dependent on the physical substrate and the concentration of fibronectin. This property is normally distributed in the cell population with a remarkably narrow standard deviation.

We are now in a position to relate binding affinities to physical detachment forces. In myogenic differentiation, myoblasts represent a proliferative, progenitor population which in response to a combination of chemical and physical signals can withdraw from the cell cycle and initiate the synthesis and assembly of the array of specific proteins which compose the contractile apparatus. The experimental evidence implicates the interaction between a5b1 integrin and fibronectin as the critical adhesive receptor interaction necessary for the differentiation to progress. Since antibodies can substitute for the natural ligand only if they are cross-linked to a solid substrate, the physical component of this process appears to involve an anchoring function. However, simple adhesion to the substrate is not sufficient for the cells to differentiate suggesting that it is the strength of the attachment which is critical. The progression of normally adherent cells through the cell cycle requires that the cells attach to a solid substrate using specific adhesion receptors. Again, for fibroblasts and related cell types, it appears that the a5b1 integrin is the critical interaction. Normal fibroblasts in suspension or under conditions of limited attachment appear to be arrested in a G0-like state similar to cells which are provided a substrate but limited growth factor. In this system also adhesion is not a simple parameter because adhesion to fibronectin is also an element in maintaining cells in the G0 state. Analysis of these paradoxes is fundamental to our understanding of how physical information is interpreted by cells.