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Epidermal growth factor and its receptor, basic fibroblast growth factor, transforming growth factor beta-1, and
1Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas 75235-9057.
Abstract:
The authors tried to determine whether human corneal endothelial cells in primary culture synthesize messenger RNA (mRNA) coding for epidermal growth factor (EGF), EGF receptor, basic fibroblast growth factor (FGFb), transforming growth factor beta-1 (TGFb1), and interleukin-1 alpha (IL-1 alpha). Oligodeoxythymidine-primed complementary DNA (cDNA) was generated from total cellular RNA extracted from eight independent primary corneal endothelial cell cultures. Four of these cultures, maintained 18-51 days, had obvious increases in cell numbers and mass over the 2 weeks before RNA extraction and were populated primarily with cells that were small, uniform, and mononuclear (proliferative cultures). The morphology of the cells in other four cultures, maintained 47-78 days, was predominantly large, irregular, vacuolated, and occasionally multinucleated. These cells were identical to senescent cells found in previous studies, and the cell number did not increase in these cultures over the 2 weeks preceding RNA extraction (senescent cultures). The polymerase chain reaction (PCR) was used to amplify the growth factors (EGF, FGFb, TGFb1, and IL-1 alpha), EGF receptor, and beta actin sequences from each of the cDNA samples. The EGF receptor, FGFb, and beta actin mRNAs were present in all eight cDNA samples. The EGF mRNAs were detected by PCR alone in four of the samples from proliferative cultures, TGFb1 mRNAs in three, and IL-1 alpha mRNAs in three. In the samples from senescent cultures, 0, 1, and 0 mRNAs were detected, respectively. Southern blots of the PCR products were probed with oligonucleotides complementary to sequences in each of the amplified products. This technique showed that the appropriately sized amplification products were specific.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Human corneal endothelial cells synthesize growth factor mRNAs, particularly in proliferative states. Senescent cells show significantly reduced or absent expression of these key signaling molecules.
Area of Science:
- Cell Biology
- Molecular Biology
- Ophthalmology
Background:
- Human corneal endothelial cells (HCECs) play a crucial role in maintaining corneal clarity.
- Understanding the molecular mechanisms regulating HCEC function, including growth factor signaling, is vital for ocular health.
- Primary HCEC cultures provide a model to study cellular processes like proliferation and senescence.
Purpose of the Study:
- To investigate the synthesis of messenger RNA (mRNA) for epidermal growth factor (EGF), EGF receptor, basic fibroblast growth factor (FGFb), transforming growth factor beta-1 (TGFb1), and interleukin-1 alpha (IL-1 alpha) in primary HCECs.
- To compare the expression profiles of these mRNAs in proliferative and senescent HCEC cultures.
- To determine the presence of specific growth factor and receptor mRNAs in HCECs.
Main Methods:
- Extraction of total cellular RNA from primary HCEC cultures.
- Generation of complementary DNA (cDNA) using oligodeoxythymidine priming.
- Polymerase chain reaction (PCR) amplification of target gene sequences (EGF, EGF receptor, FGFb, TGFb1, IL-1 alpha, beta actin).
- Southern blot analysis for confirmation of PCR product specificity.
Main Results:
- EGF receptor, FGFb, and beta actin mRNAs were detected in all eight HCEC cultures (proliferative and senescent).
- EGF, TGFb1, and IL-1 alpha mRNAs were detected in proliferative cultures but were significantly reduced or absent in senescent cultures.
- Specifically, EGF mRNA was found in 4/4 proliferative cultures, TGFb1 in 3/4, and IL-1 alpha in 3/4, while senescent cultures showed 0, 1, and 0 detections, respectively.
Conclusions:
- Primary human corneal endothelial cells synthesize mRNAs for EGF, EGF receptor, FGFb, TGFb1, and IL-1 alpha.
- The expression of EGF, TGFb1, and IL-1 alpha mRNAs is significantly diminished or lost in senescent HCEC cultures compared to proliferative ones.
- These findings suggest a differential regulation of growth factor gene expression during HCEC aging and senescence.