Mammalian TGF-beta Isoforms Research Articles

PCR detection of cytokines in normal human and pagetic osteoblast-like cells

We investigated the expression of cytokine transcripts in osteoblast-like cells derived from explants of pagetic and normal bone. A reverse transcription-linked PCR was used that allowed the simultaneous analysis of a range of cytokines. Normal osteoblast-like cells were found to contain the transcripts for IL-1 beta, IL-6, and TGF-beta 1. For the first time we detected in bone cells the two other mammalian isoforms of TGF-beta, beta 2, and beta 3. Furthermore, we have also identified mRNA for IL-3 and the novel chemotactic factor, IL-8. Using this sensitive technique it was not possible to detect mRNA for IL-1 alpha, IL-2, IL-4, IL-5, IL-7, TNF-alpha, or interferon-gamma. The human osteosarcoma cell line Saos-2 also showed a similar pattern of expression of these cytokines to primary osteoblast-like cells, with the exception that TNF-alpha was also identified. Cells isolated from pagetic bone showed essentially the same profile of cytokine expression as normal bone except that TNF-alpha was also detected in two of four samples. The cytokine profile of successive populations of cells harvested from one explant culture at 9, 22, and 57 days showed a consistent pattern of cytokine expression, demonstrating the phenotypic stability of the osteoblast-like cells in long-term cultures.

A dominant-negative receptor for type beta transforming growth factors created by deletion of the kinase domain

To prove the postulated role of type beta transforming growth factors (TGF beta) in cardiac development and other events, specific inhibitors of TGF beta signal transduction are needed. We truncated the type II TGF beta receptor cDNA (delta kT beta RII), to delete the predicted serine/threonine kinase cytoplasmic domain. delta kT beta RII was co-transfected into neonatal cardiac myocytes, together with reporter constructs for two cardiac-restricted genes that are regulated antithetically by TGF beta. delta kT beta RII impaired activation of the skeletal alpha-actin promoter by TGF beta 1, -2, and -3 and, conversely, impaired TGF beta inhibition of alpha-myosin heavy chain transcription. Thus, a kinase-defective T beta RII blocks signaling by all three mammalian TGF beta isoforms, and can disrupt both positive and negative control of transcription by TGF beta.

The role of TGF-beta s in mammalian development and neoplasia.

To date, three mammalian TGF-beta isoforms have been identified, each encoded by different genetic loci. Through each is very similar in primary amino acid structure, there are clear differences both in the mature bioactive peptide region and in the latency-associated peptide, which could potentially confer differential biological specificity. As one route to investigate differential biological function in vivo we have used gene specific probes for in situ hybridization studies to examine the distribution of RNA transcripts during mammalian embryogenesis. Mouse embryos from 6 to 14.5 gestational age and human embryos from 32 to 57 days post-fertilization have been probed. A general conclusion from these studies is that each TGF-beta gene has a distinct, through overlapping, pattern of transcript distribution and that this pattern, in most cases, is conserved between mouse and man. We have focused on the biological function the TGF-betas play in certain epithelia and in cardiogenesis, which will be discussed in this presentation.

Regulation of mammary growth and function by TGF-beta.

We have previously shown that TGF-beta 1 rapidly and reversibly inhibits ductal growth in vivo when administered by miniature slow-release plastic implants. A possible role for endogenous TGF-beta 1 was suggested by the observation that the normal gland displayed substantial, developmentally regulated levels of TGF-beta 1 transcripts and protein. These studies have now been extended to include the other two mammalian TGF-beta isoforms. When tested with slow-release plastic implants, TGF-beta 2 and TGF-beta 3 also caused disappearance of the proliferating mammary stem cell layer, with rapid involution of ductal end buds and cessation of glandular growth. None of the isoforms was active in inhibiting alveolar morphogenesis. We conclude that under the conditions of these tests, the three mammalian isoforms are functionally equivalent. However, striking differences in patterns of gene expression and in the distribution of immunoreactive peptides suggest that TGF-beta 2 was expressed only at low levels, and mainly during pregnancy. TGF-beta 3 was expressed in ductal stroma and epithelium, and was the only isoform detected in myoepithelial cells. Developing alveolar tissue and its associated ducts displayed striking TGF-beta 3 gene expression and immunostaining, which were greatly reduced during lactation. We are now investigating the possibility that the observed high levels of TGF-beta expression in pregnancy, particularly of TGF-beta 3, and the absence of substantial expression of any isoform during lactation, may indicate a role for the TGF-beta in regulating functional differentiation or the onset of milk secretion.