Final mechanism

• Cell migration needed for:

  Development, Angiogenesis, Wound Repair, Immune Responses
• Cell Layer is Wounded

  1. Cells along the edge of the wound begin to migrate

  2. Growth factors are released

  3. Migration is Complete When The Wound is Closed
• Plasmin

  A protease which degrades ECM proteins and decreases cell adhesion
• Abnormal cell migration is seen in:

  • Metastasis
  • Wound healing and immune system disorders
  • Defects in development and angiogenesis
• Plasminogen Activator Inhibitor Type 1 (PAI-1) binds to u-PA an inhibits its activity
• When PAI-1 bind u-PA, Plasminogen is not converted to plasmin and cell adhesion is maintained
• Elevated PAI-1 levels:

  • Thrombocytopenia
  • Increased risk of cancer
  • Thrombosis
  • Obesity
  • Diabetes
  • Atherosclerosis
• Does TGF-B induce cell migration

  1. Proteins differently expressed in response to TGF-B
  2. PAI-1 mRNA is also induced and how induction occurs
  3. Regions in PAI-1 promoter that may be involved in activation
  4. What proteins are bound to PAI-1 promoter site
  5. E-box in PAI-1 promoter plays role in activating gene in response to TGF-B
• Protocol used to determine if there are any proteins differently expressed in response to stimulus?

  SDS-PAGE, 2D Gel Electrophoresis, Western Blot
• What protocols used to see whether mRNA is also induced by stimulus and how that occurs?

  Northern blot, mRNA half life, Nuclear Run Off
• How to see if there are any regions in the promoter of interest that may be involved in the activation?

  DNA sequencing, EMSA (mobility shifts)
• How to determine what protein(s) are bound to the promoter site of interest?

  Supershift, Depletion, Using E-Box (or AP-1) site as probe
• How to see whether the E-box in promoter plays a role in activating gene in response to stimulus?

  Reporter (CAT/LUC) Assays using promoter of interest with WT/Mutated E-box
• Once a signaling pathway has been identified the next step is to see if this pathway is actually involved

  • can use inhibitors, antisense, and over expression
• Example pathway induced by TGF-B : Ras

  1. Is Ras involved in activating MEK-1/2 and ERK-1/2 in response to TGF-B?

  Perform Western for P-ERK-1/2 by inhibit/OE Ras and see if ERK-1/2 is activated (phosphorylated)
• Example pathway induced by TGF-B : Ras

  2. Is Ras involved in activating USF-1 binding to an E-Box in response to TGF-B?

  Perform Mobility Shift and Inhibit/OE Ras and see if USF-1 still binds to E-box
• Example pathway induced by TGF-B : Ras

  3. Is Ras involved in activating PAI-1 protein levels in response to TGF-B?

  Perform Western Blot by Inhibit/OE Ras and see if PAI-1 protein levels still increase
• Western Blot or EMSA Vector for Ras

  • ORI
  • Eukaryotic and Prokaryotic Ab resistance gene
  • CMV or SV-40 promoter
  • Ras anti-sense or over-expression gene
• Promoter/Reporter Assay vector for Ras - need 2 vectors

  • ORI
  • Eukaryotic and Prokaryotic Ab resistance gene
  • Ras promoter/enhancer with WT E-Box site/mutated E-Box site
  • Luciferase or CAT
• When it says to inhibit or over express a gene, use antisense or over-expression vectors
• Example pathway induced by TGF-B : Ras

  4. Is Ras involved in inducing cell migration in response to TGF-B?

  Measure cell migration by Inhibit/OE Ras and see if cell migration has changed
• Example pathway induced by TGF-B : MEK-1/2 and ERK-1/2

  1. Is MEK-1/2 involved in activating ERK-1/2 in response to TGF-B?

  Perform Western Blot for P-ERK-1/2 and Inhibit/OE MEK-1/2 and see if ERK-1/2 is activated (phosphoylated)
• MEK-1/2 inhibitors that can be used to determine activation: PD98059 and U0126
• Example pathway induced by TGF-B : MEK-1/2 and ERK-1/2

  2. Is MEK-1/2/ERK-1/2 involved in activating USF-1 binding to an E-Box in response to TGF-B?

  Perform Mobility Shift using E-box as probe and Inhibit/OE MEK-1/2/ERK-1/2 and see if USF-1 still binds to E-box
• Example pathway induced by TGF-B : MEK-1/2 and ERK-1/2

  3. Is MEK-1/2/ERK-1/2 involved in activating PAI-1 protein levels in response to TGF-B?

  Perform Western Blot for PAI-1 and inhibit/OE MEK-1/2/ERK-1/2 and see if PAI-1 levels still increase
• Example pathway induced by TGF-B : MEK-1/2 and ERK-1/2

  4. Is MEK-1/2/ERK-1/2 involved in inducing cell migration in response to TGF-B?

  Measure cell migration by inhibit/OE MEK-1/2/ERK-1/2 and see if cell migration has changed
• Example pathway induced by TGF-B : USF-1

  1. Is USF-1 involved in activating USF-1 binding to an E-box in response to TGF-B?

  Perform Mobility Shift using e-box as a probe and inhibit/OE USF-1 and see if USF-1 still binds to the E-box
• Example pathway induced by TGF-B : USF-1

  2. Is USF-1 involved in activating PAI-1 protein levels in response to TGF-B?

  Perform Western Blot for PAI-1 and inhibit/OE USF-1 and see if PAI-1 protein levels still increase
• Example pathway induced by TGF-B : USF-1

  3. Is USF-1 involved in inducing cell migration in response to TGF-B?

  Measure cell migration by inhibit/OE USF-1 and see if cell migration has changed
• Example pathway induced by TGF-B : PAI-1

  1. Is PAI-1 involved in inducing cell migration in response to TGF-B?

  Measure cell migration by inhibit/OE PAI-1 and see if cell migration has changed
• PAI-1 is a prognostic indicator for: Breast, Pancreatic, Bladder, and Colorectal cancer; Type II diabetes
• Uses for PAI-1 in therapy:

  • Antagonist for intravasculcar thrombosis
  • Thiazolidinediones (TZDs)
  • Small molecule PAI-1 inhibitors
• Thiazolidinediones (TZDs) decrease PAI-1 levels in adipose tissues which can improve insulin resistance and decrease cardiovascular risk
• Small molecule PAI-1 inhibitors - studying treatments in: ovarian, cervical, and bladder cancer; Lewis lung carcinoma; Intestinal polyps
• Protocols used to determine cell proliferation: flow cytometry, mitotic shake, hemocytometer, BrDu Staining, 3H-Thymidine Uptake