Master’s-Level Cell Biology & Advanced Molecular Biology Notes

1. Definition

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteolytic enzymes that cleave extracellular-matrix (ECM) proteins and other extracellular or cell-associated substrates.

They are major regulators of:

• ECM turnover
• Tissue remodeling
• Cell migration
• Wound healing
• Angiogenesis
• Inflammation
• Development
• Cancer invasion
• Fibrosis

MMPs are not simply “matrix-destroying enzymes”; they are regulated molecular remodeling enzymes that alter the biochemical and mechanical information contained within the ECM.

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2. Basic Reaction

At a simplified level:

ECM protein
     ↓
   MMP
     ↓
Proteolytic cleavage
     ↓
ECM fragments
     ↓
Altered matrix architecture
+
New signaling molecules

MMP-mediated cleavage can therefore alter both structure and cell signaling.

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3. Why Are They Called Metalloproteinases?

The term has three components:

Matrix

Many substrates are components of the extracellular matrix.

Metallo

Their catalytic activity requires a metal ion, primarily Zn²⁺.

Proteinase

They hydrolyze peptide bonds in proteins.

Thus:

Matrix + Metalloproteinase = MMP

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4. MMPs Are Part of a Larger Protease Network

ECM degradation involves several protease families.

                 ECM PROTEOLYSIS
                       │
       ┌───────────────┼────────────────┐
       ↓               ↓                ↓
      MMPs            ADAMs           ADAMTS
       │               │                │
   ECM cleavage   Ectodomain       ECM/proteoglycan
                  shedding           processing

Other proteases, including serine proteases such as plasmin, can also participate in ECM remodeling.

---

5. Human MMP Family

Humans have approximately 23 known MMP genes.

They are classified according to:

• Structure
• Cellular localization
• Substrate preference
• Functional characteristics

Major groups include:

1. Collagenases
2. Gelatinases
3. Stromelysins
4. Matrilysins
5. Membrane-type MMPs
6. Other MMPs

---

6. General MMP Structure

Many classical MMPs have several domains:

N-terminus
   │
   ↓
Signal peptide
   │
   ↓
Propeptide
   │
   ↓
Catalytic domain
   │
   ↓
Hinge region
   │
   ↓
Hemopexin domain
   │
   ↓
C-terminus

Not every MMP has exactly this organization.

---

7. Signal Peptide

Secreted MMPs generally possess an N-terminal signal peptide.

It directs the newly synthesized protein into the:

rough endoplasmic reticulum → secretory pathway

MMP mRNA
   ↓
Ribosome
   ↓
Signal peptide
   ↓
Rough ER
   ↓
Golgi
   ↓
Extracellular space

---

8. Propeptide Domain

Most MMPs are synthesized as inactive zymogens, called:

Pro-MMPs

The propeptide maintains the enzyme in an inactive state.

A conserved region contains a cysteine that interacts with the catalytic zinc.

This is often described as the:

“cysteine switch”

---

9. Cysteine-Switch Mechanism

Simplified:

             PRO-MMP
                │
        Cys residue binds
          catalytic Zn²⁺
                │
                ↓
        Catalytic site blocked
                │
                ↓
             INACTIVE

Activation disrupts this interaction.

Pro-MMP
   ↓
Propeptide cleavage / structural disruption
   ↓
Cysteine-Zn²⁺ interaction lost
   ↓
Catalytic site exposed
   ↓
ACTIVE MMP

---

10. Catalytic Domain

The catalytic domain contains the machinery required for proteolysis.

A critical feature is the presence of:

Zn²⁺

The catalytic zinc participates in peptide-bond hydrolysis.

---

11. Hemopexin Domain

Many MMPs contain a C-terminal hemopexin-like domain.

It contributes to:

• Substrate recognition
• Protein–protein interactions
• Collagen binding
• Regulation of activity

Some MMPs lack a conventional hemopexin domain.

---

12. MMP Activation

MMPs are tightly regulated because uncontrolled proteolysis would damage tissues.

General activation:

Inactive pro-MMP
       ↓
Extracellular activation
       ↓
Propeptide disruption/removal
       ↓
Active MMP
       ↓
ECM cleavage

Activation may involve:

• Other proteases
• Proteolytic processing
• Cell-surface activation mechanisms
• Chemical/oxidative modifications in some contexts

---

13. MMP Cascade

MMPs can activate other MMPs.

Pro-MMP
   ↓
Active MMP
   ↓
Activation of other pro-MMPs
   ↓
Amplified proteolysis

Therefore, MMP activity can behave as a proteolytic cascade.

---

14. Major MMP Classes

Class	Examples	Major role
Collagenases	MMP-1, MMP-8, MMP-13	Fibrillar collagen cleavage
Gelatinases	MMP-2, MMP-9	Gelatin and basement-membrane components
Stromelysins	MMP-3, MMP-10	Broad ECM remodeling
Matrilysins	MMP-7, MMP-26	Small ECM proteins and other substrates
Membrane-type MMPs	MMP-14, MMP-15, MMP-16, MMP-17	Cell-surface/pericellular proteolysis

---

15. Collagenases

Important collagenases include:

• MMP-1
• MMP-8
• MMP-13

They can cleave fibrillar collagens.

Major targets include:

• Type I collagen
• Type II collagen
• Type III collagen

depending on the enzyme and biological context.

---

16. MMP-1

MMP-1 = Interstitial collagenase

Important in:

• Wound healing
• Inflammation
• Tissue remodeling
• Cancer
• Arthritis

It cleaves fibrillar collagen and generates fragments that can subsequently be degraded by other proteolytic processes.

---

17. MMP-8

MMP-8 is also known as:

Neutrophil collagenase

It is associated particularly with neutrophils and inflammatory environments.

It contributes to:

• Collagen turnover
• Inflammation
• Wound repair

---

18. MMP-13

MMP-13 = Collagenase-3

It is particularly important in:

• Cartilage remodeling
• Bone remodeling
• Development
• Arthritis
• Tumor-associated matrix remodeling

---

19. Gelatinases

The major gelatinases are:

• MMP-2
• MMP-9

They degrade:

• Gelatin
• Denatured collagen
• Type IV collagen
• Other basement-membrane-associated substrates

---

20. MMP-2

MMP-2 = Gelatinase A

MMP-2 participates in:

• Basement-membrane remodeling
• Angiogenesis
• Cell migration
• Development
• Cancer invasion

It can interact with cell-surface components and is particularly important in pericellular matrix remodeling.

---

21. MMP-9

MMP-9 = Gelatinase B

MMP-9 is produced by several cell types, including:

• Neutrophils
• Macrophages
• Some tumor cells
• Other inflammatory and stromal cells

It participates in:

• Inflammation
• Angiogenesis
• Wound healing
• Basement-membrane remodeling
• Tumor invasion

---

22. Stromelysins

Important stromelysins include:

• MMP-3
• MMP-10

They have broad substrate specificity.

They can degrade or process:

• Proteoglycans
• Fibronectin
• Laminin
• Other ECM proteins

They can also participate in activation of other MMPs.

---

23. MMP-3

MMP-3 = Stromelysin-1

It is important because it:

• Degrades several ECM components
• Participates in tissue remodeling
• Can activate other MMPs

Thus MMP-3 can contribute to proteolytic amplification.

---

24. Matrilysins

Important examples:

• MMP-7
• MMP-26

Matrilysins are relatively small MMPs.

They lack the conventional hemopexin domain found in many other MMPs.

---

25. MMP-7

MMP-7 is involved in:

• ECM remodeling
• Epithelial biology
• Inflammation
• Wound healing
• Tumor progression

Importantly, MMP-7 can process substrates beyond classical ECM proteins.

---

26. Membrane-Type MMPs

Some MMPs are associated with the cell membrane.

Examples include:

• MMP-14
• MMP-15
• MMP-16
• MMP-17

These are important for pericellular proteolysis.

---

27. MMP-14

MMP-14 = MT1-MMP

MMP-14 is a major membrane-associated MMP.

It participates in:

• Cell migration
• Invasion
• ECM remodeling
• Angiogenesis
• Activation of MMP-2

A simplified pathway:

Cell membrane
      │
   MMP-14
      │
      ↓
Pro-MMP-2
      ↓
Active MMP-2
      ↓
ECM remodeling

---

28. Pericellular Proteolysis

MMPs can concentrate proteolysis near the cell surface.

             ECM
─────────────────────────
      ↑           ↑
    MMP-2       MMP-9
      ↑
   MMP-14
      │
┌─────┴─────┐
│   CELL    │
└───────────┘

This allows cells to create a locally remodeled pathway through the ECM.

---

29. MMPs and Cell Migration

For a cell to migrate through dense ECM, matrix barriers may need to be modified.

Cell
 ↓
Integrin adhesion
 ↓
MMP localization
 ↓
ECM proteolysis
 ↓
Matrix remodeling
 ↓
Cell movement

This is important during:

• Development
• Immune-cell migration
• Wound healing
• Cancer invasion

---

30. MMPs and Integrins

Integrins and MMPs cooperate.

Integrins

→ attach cells to ECM

MMPs

→ modify ECM

Together:

ECM
 ↓
Integrin
 ↓
Cell adhesion
 ↓
MMP localization
 ↓
ECM remodeling
 ↓
Cell migration

This creates coordinated adhesion–proteolysis cycles.

---

31. MMPs and Focal Adhesions

MMP activity can alter the ECM surrounding focal adhesions.

ECM
 ↓
Integrin
 ↓
Focal adhesion
 ↓
Actin cytoskeleton

MMP-mediated ECM modification can change:

• Integrin ligand availability
• Adhesion strength
• Cytoskeletal tension
• Cell migration

---

32. MMPs and Angiogenesis

Angiogenesis requires endothelial cells to:

1. Activate
2. Degrade/remodel surrounding matrix
3. Migrate
4. Proliferate
5. Form vascular structures

MMPs participate in several of these steps.

Angiogenic stimulus
       ↓
Endothelial activation
       ↓
MMP activity
       ↓
ECM remodeling
       ↓
Endothelial migration
       ↓
New vessel formation

---

33. MMPs in Wound Healing

MMP activity changes during different stages of repair.

Injury
  ↓
Inflammation
  ↓
MMP activation
  ↓
Removal/remodeling of damaged ECM
  ↓
Cell migration
  ↓
New matrix deposition
  ↓
Matrix maturation

MMPs therefore participate in both matrix removal and matrix reorganization.

---

34. MMPs in Inflammation

Inflammatory cells can produce MMPs.

MMPs can:

• Remodel ECM
• Facilitate leukocyte migration
• Process cytokines
• Process chemokines
• Alter cell-surface receptors

Therefore, MMPs can regulate inflammation at multiple levels.

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35. MMPs and Cytokine Processing

MMPs can sometimes modify signaling molecules through proteolytic processing.

Thus:

MMP
 ↓
Cytokine / receptor processing
 ↓
Altered signaling

This demonstrates that MMPs have functions beyond simple ECM degradation.

---

36. MMPs and Growth Factors

ECM can act as a reservoir for growth factors.

MMP-mediated remodeling can alter their availability.

ECM-bound growth factor
          ↓
      MMP activity
          ↓
Release / redistribution
          ↓
Growth-factor receptor
          ↓
Cell signaling

---

37. MMPs and Cancer

MMPs are frequently associated with tumor progression, but their role is context-dependent and more complex than simply “MMPs cause cancer.”

They may influence:

• ECM invasion
• Angiogenesis
• Tumor-cell migration
• Growth-factor availability
• Immune-cell recruitment
• Metastatic niche formation

---

38. Cancer Invasion

A simplified model:

Primary tumor
     ↓
ECM remodeling
     ↓
Basement-membrane disruption
     ↓
Tumor-cell migration
     ↓
Stromal invasion
     ↓
Intravasation
     ↓
Metastasis

MMPs can contribute to several of these steps.

---

39. MMPs and Basement Membrane

Basement membranes contain:

• Type IV collagen
• Laminins
• Nidogen
• Proteoglycans

MMPs can modify components of this matrix.

Basement membrane
       ↓
MMP activity
       ↓
Matrix remodeling
       ↓
Altered barrier properties

---

40. MMPs in Fibrosis

MMPs have complex roles in fibrosis.

They can:

• Degrade existing matrix
• Process ECM components
• Release signaling molecules
• Alter fibroblast behavior

Therefore, fibrosis cannot simply be explained as:

“low MMP = fibrosis.”

Different MMPs can have different effects depending on:

• Tissue
• Timing
• Cellular source
• Substrate
• Disease stage

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41. MMP–TIMP Balance

One of the most important concepts:

             MMP–TIMP BALANCE

      MMP activity       TIMP activity
           │                   │
           ↓                   ↓
      ECM degradation      MMP inhibition
           │                   │
           └─────────┬─────────┘
                     ↓
              ECM homeostasis

Excessive MMP activity can cause matrix destruction.

Excessive inhibition or excessive matrix synthesis can contribute to matrix accumulation.

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42. TIMP-1

TIMP-1 is an endogenous inhibitor of several MMPs.

It can bind active MMPs and limit proteolytic activity.

TIMP-1 also has biological effects that extend beyond simple MMP inhibition.

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43. TIMP-2

TIMP-2 inhibits several MMPs and has an important relationship with MMP-14/MT1-MMP.

Interestingly, TIMP-2 participates in the molecular organization required for pro-MMP-2 activation.

Thus:

A TIMP can participate in MMP activation machinery while also inhibiting MMP activity.

This is an important advanced concept.

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44. MMP Activation and Cell Surface

A simplified MT1-MMP/MMP-2 system:

                 CELL SURFACE

             MMP-14      MMP-14
                │           │
                └─────┬─────┘
                      │
                    TIMP-2
                      │
                 Pro-MMP-2
                      ↓
                 Active MMP-2
                      ↓
                    ECM

The precise molecular arrangement is more complex, but this illustrates the principle.

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45. MMPs and Mechanotransduction

ECM mechanics can influence protease activity.

Conversely, MMP-mediated degradation can alter matrix mechanics.

Therefore:

Matrix mechanics
       ↓
Cell signaling
       ↓
MMP expression/activity
       ↓
ECM remodeling
       ↓
New matrix mechanics

This creates a mechanochemical feedback loop.

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46. Regulation of MMP Expression

MMP expression can be regulated at multiple levels.

Transcriptional regulation

Important pathways include:

• AP-1
• NF-κB
• MAPK signaling

Post-transcriptional regulation

• mRNA stability
• microRNAs

Translational regulation

Changes in protein synthesis.

Post-translational regulation

• Zymogen activation
• Inhibitor binding
• Cellular localization

Stimulus
   ↓
Signal pathway
   ↓
Transcription factor
   ↓
MMP gene transcription
   ↓
Pro-MMP synthesis
   ↓
Activation
   ↓
ECM remodeling

---

47. Inflammatory Regulation

Inflammatory signals can increase MMP expression.

Examples include signaling involving:

• TNF
• IL-1
• TLR pathways
• NF-κB
• MAPKs

Inflammatory stimulus
       ↓
NF-κB / AP-1
       ↓
MMP transcription
       ↓
Pro-MMP production
       ↓
Activation

---

48. MMPs and Reactive Oxygen Species

Reactive oxygen species can influence extracellular protease systems.

Oxidative conditions may alter:

• MMP expression
• MMP activation
• ECM susceptibility

This connects:

oxidative stress ↔ inflammation ↔ ECM remodeling

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49. MMPs and ECM Fragments

ECM degradation can produce bioactive fragments.

ECM protein
     ↓
MMP cleavage
     ↓
ECM fragment
     ↓
Receptor interaction
     ↓
Cell signaling

These fragments can influence:

• Migration
• Inflammation
• Angiogenesis
• Cell proliferation

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50. MMPs in Development

MMPs participate in:

• Embryonic tissue remodeling
• Cell migration
• Organ development
• Vascular development
• Skeletal development

Their activity is tightly spatially and temporally controlled.

---

51. MMPs and Bone Remodeling

MMPs contribute to:

• Collagen turnover
• Osteoblast/osteoclast interactions
• Matrix processing

They complement the activity of other bone-remodeling proteases.

---

52. MMPs and Cartilage

Cartilage contains abundant:

• Type II collagen
• Aggrecan

MMPs and ADAMTS enzymes participate in cartilage matrix turnover.

Dysregulated proteolysis contributes to cartilage destruction in osteoarthritis.

---

53. MMPs in the Nervous System

MMPs can influence:

• Neural development
• Synaptic plasticity
• Blood–brain barrier remodeling
• Neuroinflammation
• Neural injury responses

Their effects depend strongly on timing and cellular context.

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54. MMPs and Blood–Brain Barrier

MMP activity can modify components of the neurovascular unit and basement membrane.

Excessive MMP activity may contribute to barrier disruption under certain pathological conditions.

MMP activity
    ↓
Basement-membrane / junctional protein remodeling
    ↓
Altered barrier integrity

---

55. Therapeutic Targeting of MMPs

Because excessive MMP activity contributes to several diseases, MMP inhibition has been extensively investigated.

However, broad MMP inhibition has proved challenging because:

• MMPs have overlapping substrates
• Different MMPs have different functions
• Some MMPs are protective
• MMPs participate in normal tissue repair
• Timing is critical

Therefore, modern approaches emphasize selective and context-specific inhibition.

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56. Why Broad MMP Inhibition Can Fail

A simplistic strategy:

Block all MMPs
       ↓
Less ECM degradation

But physiologically:

MMPs
 ├── Pathological effects
 └── Normal repair / remodeling

Blocking all MMP activity can therefore produce undesirable effects.

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57. Modern View of MMP Biology

The older concept:

MMP = ECM destruction

The modern concept:

MMP = regulated extracellular proteolytic signaling and matrix-remodeling system

MMPs can:

• Remove matrix
• Reshape matrix
• Release matrix-bound factors
• Process cytokines
• Process receptors
• Generate matrikines
• Regulate migration
• Influence angiogenesis

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58. Integrated Molecular Pathway

                 INFLAMMATION
                      │
                      ↓
               NF-κB / AP-1
                      │
                      ↓
               MMP transcription
                      │
                      ↓
                  Pro-MMP
                      │
              activation signals
                      ↓
                 ACTIVE MMP
                      │
       ┌──────────────┼──────────────┐
       ↓              ↓              ↓
     ECM          Growth factors   Receptors
   degradation      release/       processing
                    change
       │              │              │
       └──────────────┼──────────────┘
                      ↓
                Cell behavior
                      ↓
       Migration / angiogenesis /
       inflammation / remodeling

---

59. MMPs vs TIMPs

Feature	MMPs	TIMPs
Main function	Proteolysis	Inhibit many MMPs
Major location	Extracellular/pericellular	Extracellular
Major effect	ECM remodeling	Restrain proteolysis
Regulation	Complex	Complex
Role in cancer	Context-dependent	Context-dependent
Role in repair	Important	Important
Major concept	Matrix modification	Protease control

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60. High-Yield MMP Table

MMP	Common name	Major significance
MMP-1	Interstitial collagenase	Fibrillar collagen
MMP-2	Gelatinase A	Basement-membrane/pericellular remodeling
MMP-3	Stromelysin-1	Broad ECM remodeling; MMP activation
MMP-7	Matrilysin	Small ECM proteins, epithelial biology
MMP-8	Neutrophil collagenase	Inflammation/collagen turnover
MMP-9	Gelatinase B	Inflammation, angiogenesis, BM remodeling
MMP-13	Collagenase-3	Cartilage/bone remodeling
MMP-14	MT1-MMP	Cell-surface proteolysis; MMP-2 activation

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61. Examination Answer

Matrix Metalloproteinases

Matrix metalloproteinases are a family of zinc-dependent proteolytic enzymes that participate in extracellular-matrix remodeling and the processing of numerous extracellular and cell-associated substrates. Humans possess approximately 23 MMP genes.

Most MMPs are synthesized as inactive proenzymes (zymogens) containing a propeptide that maintains catalytic inactivity through a cysteine-switch mechanism involving the catalytic zinc ion. Activation involves disruption or removal of the propeptide, exposing the catalytic site.

MMPs are classified into collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs and other groups. Important examples include MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13 and MMP-14.

Their activity is controlled by endogenous tissue inhibitors of metalloproteinases (TIMPs). The MMP–TIMP balance is important for maintaining ECM homeostasis.

MMPs participate in development, wound healing, angiogenesis, inflammation, cell migration, tissue regeneration and cancer progression. They also process growth factors, cytokines, receptors and other extracellular proteins, demonstrating that their functions extend beyond simple ECM degradation.

Dysregulated MMP activity contributes to pathological conditions including cancer invasion, chronic inflammation, arthritis and tissue fibrosis. Consequently, MMPs are important therapeutic targets, although broad inhibition is difficult because individual MMPs can have both pathological and physiological functions.

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62. Viva Questions

Q1. What are MMPs?
Zinc-dependent proteolytic enzymes involved in ECM remodeling and extracellular protein processing.

Q2. Why are they called metalloproteinases?
Because their catalytic activity requires a metal ion, principally Zn²⁺.

Q3. In what form are most MMPs secreted?
As inactive pro-MMP zymogens.

Q4. What is the cysteine switch?
An interaction between the propeptide cysteine and catalytic zinc that maintains MMP inactivity.

Q5. Name two collagenases.
MMP-1 and MMP-13.

Q6. Name the major gelatinases.
MMP-2 and MMP-9.

Q7. What is MMP-14?
MT1-MMP, a membrane-associated MMP involved in pericellular proteolysis and activation of pro-MMP-2.

Q8. What inhibits MMPs?
TIMPs.

Q9. What is the role of MMPs in cancer?
They can facilitate ECM remodeling, invasion, angiogenesis and altered signaling, although their effects are context-dependent.

Q10. Are MMPs always harmful?
No. They are essential for normal development, wound healing and tissue remodeling.

Q11. What is a major reason broad MMP inhibitors have limitations?
MMPs have overlapping and sometimes protective physiological functions.

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63. One-Minute Revision

                 MMPs
                  │
          Zn²⁺-dependent
             proteases
                  │
          Usually pro-MMP
                  │
        Cysteine-switch mechanism
                  │
             Activation
                  ↓
             ACTIVE MMP
                  │
       ┌──────────┼───────────┐
       ↓          ↓           ↓
   Collagen     ECM        Growth-factor
   cleavage   remodeling     processing
       │          │           │
       └──────────┼───────────┘
                  ↓
            Cell behavior
                  │
      ┌───────────┼────────────┐
      ↓           ↓            ↓
   Migration   Angiogenesis   Repair
                  │
                  ↓
           PATHOLOGICAL ROLE
                  │
        Cancer / inflammation /
             fibrosis

Must remember

MMPs = zinc-dependent extracellular proteases

Most MMPs → synthesized as inactive pro-MMPs

Cysteine switch → maintains latency

MMP-1 → collagenase

MMP-2 & MMP-9 → gelatinases

MMP-3 → stromelysin

MMP-7 → matrilysin

MMP-13 → collagenase-3

MMP-14 → MT1-MMP

TIMPs → major endogenous MMP inhibitors

MMPs → ECM remodeling + signaling + migration + angiogenesis

MMP biology is context-dependent—not simply “ECM destruction.”