Procollagen alpha 2 (I) mRNA can be induced congruent to 6-fold in primary avian tendon (PAT) cells on addition of ascorbate to the culture medium. Previously, we have shown that the induction is linear after a 12 h lag and requires a total of 60-72 h to achieve maximum levels. We have now investigated in more detail the changes that have occurred in the metabolism of procollagen mRNA in fully induced cells to account for the observed induction. Ascorbate was found to triple the rate of procollagen gene transcription. In addition, there was a stabilization of the mRNA causing the half-life to increase from 10.5 h to 20 h. The increased stability of the procollagen mRNA, however, did not correlate with its ability to bind to oligo (dT)-cellulose. Since a 3-fold change in transcription rates and a 2-fold increase in half-life would account for the 6-fold overall increase in procollagen mRNA levels, we conclude that these are the primary alterations caused by ascorbate addition that give rise to the specific increase in procollagen mRNA.

In an adequate environment, primar avian tendon cells are capable of retaining both the full expression of differentiated function and a correct morphological orientation for I'week in culture. Af-high density-and-in thepresence of ascorbate; they are fully stabilized in that they devote 25-30% of their total protein synthesis to collagen, a level comparable to that in tendon cells in ovo. However, either at low density or in medium without ascorbate, they synthesize collagen at only a third of this level. If plated on a collagen matrix, these cells will orient themselves in a manner similar to that of tendon cells in vivo. Furthermore, they are capable of fully modulating the percentage of collagen synthesis upon addition or removal of ascorbate and serum. The variation in the percentage of collagen produced is a result of alterations in collagen synthesis rather than of changes in total protein synthesis or hydroxylati6n of proline-in collagen. Primary avian tendon cells, therefore, provide a suitable model for understanding the stability of tie differentiated state, the mechanism of action of ascorbate, and the regulation of collagen biosyn-Understanding what regulates gene expression at cellular and molecular levels requires a rigorously defined and controlled environment. Despite efforts of the cell biologists for the past 70 years, most cells that are removed from the organism and placed in culture lose their ability to remain differentiated (1)(2)(3)(4)(5). One conclusion that can be drawn from this universal phenomenon is that very few, if any, tissue-specific functions are "constitutive." That is, despite the fact that synthesis of differentiated products is stable in vivo, its continued expression relies on factors that are no longer present in culture. To study function in cells in culture, two approaches are therefore possible. One is to allow the cells to adapt to and reach an equilibrium with their new environment, with invariable loss of qualitative and quantitative expression of function. This is the approach traditionally taken in the past. Alternatively, the culture environment can be modified to maintain the differentiated state of the cell. This latter approach, initiated by Schwarz et al. (5, 6) for avian tendon cells, has been pursued in this research in order to achieve a culture system that more closely resembles the in vivo state of cells in terms of collagen biosynthesis.The previous research on collagen synthesis, using "fibroblasts" that had adapted to standard cell culture conditions, has been confusing for several reasons. First, most cells in the body have the capability to synthesize and secrete some collagen; it is the type and the quantity that vary (7-9). Therefore, the level of differentiation retained by the cell in culture can only be delineated with a knowledge of the in vivo origin of the fibroblast. This information is unavailable for almost all cell lines used currently. Second, in the early research on collagen synthesis by fibroblasts in culture, the assay used...

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BackgroundScaling protein production seems like a simple perturbation of transcriptional control. However, when embryonic tendon fibroblasts have to produce >50% procollagen and secrete it from the cell 4 times faster than the average protein, this taxes the cellular machinery and requires a fresh look at how the pathway is controlled. Ascorbate, a reducing agent, can stimulate procollagen production 6-fold. Procollagen mRNA levels goes up 6-fold but requires 3 days for the cell to accomplish this task. Secretion rates, the last cellular step in the process, also goes up 6-fold but this occurs in <1 h. What regulatory scheme is consistent with these properties?Scope of this reviewThis review focuses on fibroblasts that make high levels of procollagen (type I) and how they regulate the collagen pathway. Data from many different labs are relevant to this problem but it is hard to see the bigger picture from a large number of small studies. This review aims to consolidate this data into a coherent model and this requires solutions to some controversies and postulating potential mechanisms where the details are still missing.Major conclusionsIn high collagen producing cells, the pathway is controlled by post-transcriptional regulation. This requires feedback control between secretion and translation rates that is based on the helical structure of the procollagen molecule and additional tissue-specific modifications.General significanceTranscriptional control does not scale well to high protein production with rapid regulation. New paradigms lead to better understanding of collagen diseases and tendon morphogenesis.

Ascorbic acid displays the characteristics of an ideal inducer of tissue-specific function in primary avian tendon cells in culture. It is a highly specific, potent stimulator of collagen synthesis, it demonstrates slow reversible kinetics, and it has no effect on growth rate of the cultured cells. Kinetic analysis of ascorbate induction of collagen synthesis was used to determine the critical steps in this complex biosynthetic pathway. Full hydroxylation of the proline residues in collagen, although probably a necessary step for collagen induction, was in itself not sufficient for achieving either increased secretion or increased synthesis. On the other hand, an increase in secretion rate, which required both the presence of ascorbate and a high cell density, did correlate with the later stimulation in procollagen production. The process of procollagen secretion, therefore, meets the minimal requirements for the rate-limiting step. The fact that the cells maintained a large pool of intracellular procollagen despite changes in the rates of translation or secretion led us to postulate a possible feedback between the level of the internal procollagen pool and the rate of procollagen synthesis.The quantitative expression of tissue-specific function is precisely regulated within the organism, but this regulation can be readily lost when cells are cultured (2, 6, 10, 35). In primary avian tendon (PAT) cells, the ability to maintain in ovo levels of collagen synthesis (30% of total protein synthesized [11]) is directly related to the cell culture environment (35,36,37). Of the many factors that influence cells in culture through poorly understood mechanisms, in this paper we have focused on ascorbic acid for several reasons. Ascorbic acid serves as a clear example of how a small molecule can influence the protein synthetic machinery and the level of post-translational modifications in eucaryotic cells. In PAT cells, ascorbic acid shifts the level of protein synthesis devoted to collagen from 5 to 10% to 20 to 30% (35) and greatly increases the hydroxylation of proline residues in collagen. This modulation becomes especially significant since ascorbate deprivation in vivo, i.e., scurvy, is characterized by the same quantitative and qualitative response as that observed in PAT cells in culture (3).Additionally, although the general mechanism of ascorbate action appears to be straightfort Present address: The Jackson Laboratory, Bar Harbor, ME 04609.